Polyconjugates for in vivo delivery of polynucleotides

ABSTRACT

The present invention is directed to compounds, compositions, and methods useful for delivering polynucleotides or other cell-impermeable molecules to mammalian cells. Described are polyconjugates systems that incorporate targeting, anti-opsonization, anti-aggregation, and transfection activities into small biocompatible in vivo delivery vehicles. The use of multiple reversible linkages connecting component parts provides for physiologically responsive activity modulation.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/822,833 filed Aug. 18, 2006, and U.S. Provisional Application No.60/915,868 filed May 3, 2007.

BACKGROUND OF THE INVENTION

The delivery of polynucleotide and other membrane impermeable compoundsinto living cells is highly restricted by the complex membrane systemsof the cell. Drugs used in antisense and gene therapies are relativelylarge hydrophilic polymers and are frequently highly negatively chargedas well. Both of these physical characteristics preclude their directdiffusion across the cell membrane. For this reason, the major barrierto polynucleotide delivery is the delivery of the polynucleotide to thecellular interior. Numerous transfection reagents have been developed todeliver polynucleotides to cells in vitro. However, in vivo delivery ofpolynucleotides is complicated by toxicity, serum interactions, and poortargeting of transfection reagents that are effective in vitro.Transfection reagents that work well in vitro, cationic polymers andlipids, typically destabilize cell membranes and form large particles.The cationic charge of transfection reagent facilitates nucleic acidbinding as well as cell binding. Destabilization of membranesfacilitates delivery of the membrane impermeable polynucleotide across acell membrane. These properties render transfection reagents ineffectiveor toxic in vivo. Cationic charge results in interaction with serumcomponents, which causes destabilization of thepolynucleotide-transfection reagent interaction and poor bioavailabilityand targeting. Cationic charge may also lead to in vivo toxicity.Membrane activity of transfection reagent, which can be effective invitro, often leads to toxicity in vivo.

For in vivo delivery, a transfection complex (transfection reagent inassociation with the nucleic acid to be delivered) should be small, lessthan 100 nm in diameter, and preferably less than 50 nm. Even smallercomplexes, less that 20 nm or less than 10 nm would be more useful yet.Transfection complexes larger than 100 nm have very little access tocells other than blood vessel cells in vivo. In vitro complexes are alsopositively charged. This positive charge is necessary for attachment ofthe complex to the cell and for membrane fusion, destabilization ordisruption. Cationic charge on in vivo transfection complexes leads toadverse serum interactions and therefore poor bioavailability. Nearneutral or negatively charged complexes would have better in vivodistribution and targeting capabilities. However, in vitro transfectioncomplexes associate with nucleic acid via charge-charge (electrostatic)interactions. Negatively charged polymers and lipids do not interactwith negatively charged nucleic acids. Further, these electrostaticcomplexes tend to aggregate or fall apart when exposed to physiologicalsalt concentrations or serum components. Finally, transfection complexesthat are effective in vitro are often toxic in vivo. Polymers and lipidsused for transfection disrupt or destabilize cell membranes. Balancingthis activity with nucleic acid delivery is more easily attained invitro than in vivo.

While several groups have made incremental improvements towardsimproving gene delivery to cells in vivo, there remains a need for aformulation that effectively delivers a polynucleotide together with adelivery agent to a target cell without the toxicity normally associatedwith in vivo administration of transfection reagents. The presentinvention provides compositions and methods for the delivery and releaseof a polynucleotide to a cell using biologically labile conjugatedelivery systems.

SUMMARY OF THE INVENTION

In a preferred embodiment, the invention features a composition fordelivering a polynucleotide to a cell in vivo comprising a reversiblymasked membrane active polymer reversibly conjugated to apolynucleotide. The polymer is attached, via one or more firstreversible covalent linkages, to one or more masking agents and isfurther attached, via one or more second reversible covalent linkages,to one or more polynucleotides. The first and second reversible covalentlinkages may comprise reversible bonds that are cleaved under the sameor similar conditions or they may cleaved under distinct conditions,i.e. they may comprise orthogonal reversible bonds. Thepolynucleotide-polymer conjugate is administered to a mammal in apharmaceutically acceptable carrier or diluent.

In a preferred embodiment are disclosed membrane active polymerscomprising: polyvinylether random copolymers. The polyvinylethercopolymers may be synthesized from two, three, or more differentmonomers. Monomers may be selected from the list comprising: protectedamino vinyl ether such as phthalimido-containing vinyl ethers, butylvinyl ether, dodecyl vinyl ether, octadecyl vinyl ether. A preferredpolyvinylether random copolymer comprises three monomers: an aminecontaining monomer, a butyl vinyl ether, and an octadecyl vinyl ether.

In a preferred embodiment, one or more biophysical characteristics ofthe membrane active polymer are reversibly shielded or modified by amasking agent. Masking agents may be selected from the group comprisingsteric stabilizers, targeting groups and charge modifying agents. Themasking agent can improve biodistribution or targeting of thepolymer-polynucleotide conjugate by inhibiting non-specific interactionsof the polymer with serum components or non-target cells. The maskingagent can also reduce aggregation of the polymer orpolymer-polynucleotide conjugate. Masking agents containing targetinggroups can enhance cell-specific targeting or cell internalization bytargeting the conjugate system to a cell surface receptor. The maskingagent can be conjugated to the membrane active polymer prior to orsubsequence to conjugation of the polymer to a polynucleotide.

In a preferred embodiment, the polynucleotide that may be delivered tocells using the described conjugate systems may be selected from thegroup comprising: DNA, RNA, blocking polynucleotides, antisenseoligonucleotides, plasmids, expression vectors, oligonucleotides, siRNA,microRNA, mRNA, shRNA and ribozymes.

In a preferred embodiment, the masking agent(s) and thepolynucleotide(s) are covalently linked to the membrane active polymervia reversible linkages. While masking of the polymer, and attachment ofthe polynucleotide to the polymer, are important, these attachments caninterfere with transfection activity of the polymer or the activity ofthe polynucleotide. By attaching the masking agent and thepolynucleotide to the polymer via reversibly linkages that are cleavedat an appropriate time, activity is restored to the polymer and thepolynucleotide is released. Reversible covalent linkages containreversible or labile bonds which may be selected from the groupcomprising: physiologically labile bonds, cellular physiologicallylabile bonds, pH labile bonds, very pH labile bonds, extremely pH labilebonds, enzymatically cleavable bonds, and disulfide bonds. The presenceof two reversible linkages connecting the polymer to the polynucleotideand a masking agent provides for co-delivery of the polynucleotide witha delivery polymer and selective targeting and inactivation of thedelivery polymer by the masking agent. Reversibility of the linkagesprovides for release of polynucleotide from the membrane active polymerand selective activation of the membrane active polymer.

In a preferred embodiment, we describe a composition comprising: adelivery polymer covalently linked to: a) one or more targeting groups,steric stabilizers or charge modifiers via one or more reversiblelinkages; and, b) one or more polynucleotides via one or more reversiblelinkages. In one embodiment, the targeting agent, steric stabilizer, orcharge modifier reversible covalent linkage is orthogonal to thepolynucleotide reversible covalent linkage.

In a preferred embodiment, we describe a polymer conjugate system fordelivering a polynucleotide to a cell and releasing the polynucleotideinto the cell comprising: the polynucleotide reversibly conjugated to amembrane active polymer which is itself reversibly conjugated to amasking agent. The conjugation bonds may be the same or they may bedifferent. In addition, the conjugation bonds may be cleaved under thesame or different conditions.

In a preferred embodiment, we describe a polymer conjugate system fordelivering a membrane impermeable molecule to a cell and releasing themolecule in the cell. The polymer conjugate system comprises themembrane impermeable molecule reversibly linked to a membrane activepolymer wherein a plurality of masking agents are linked to the membraneactive polymer via reversible covalent bonds. Membrane active polymersmay be toxic or may not be targeted when applied in vivo. Reversibleattachment of a masking agent reversibly inhibits or alters membraneinteractions, serum interactions, cell interactions, toxicity, or chargeof the polymer. A preferred reversible covalent bond comprises: a labilebond, a physiologically labile bond or a bond cleavable under mammalianintracellular conditions. A preferred labile bond comprises a pH labilebond. A preferred pH labile bond comprises a maleamate bond. Anotherpreferred labile bond comprises a disulfide bond. Membrane impermeablemolecules include, but are not limited to: polynucleotides, proteins,antibodies, and membranes impermeable drugs.

In a preferred embodiment, a polynucleotide is attached to the polymerin the presence of an excess of polymer. The excess polymer may aid informulation of the polynucleotide-polymer conjugate. The excess polymermay reduce aggregation of the conjugate during formulation of theconjugate. The polynucleotide-polymer conjugate may be separated fromthe excess polymer prior to administration of the conjugate to the cellor organism. Alternatively, the polynucleotide-polymer conjugate may beco-administered with the excess polymer to the cell or organism. Theexcess polymer may be the same as the polymer or it may be different.

In a preferred embodiment, the polymer conjugates of the invention maybe used for in vitro delivery of a polynucleotide or other membraneimpermeable molecule. For in vitro delivery, reversible masking of thepolymer enhances transfection activity of some otherwise ineffectivepolyamines, such as polylysine. Reversibly masking can also enhance theutility of other membrane active polymers by reducing their toxicity orlimiting their membrane disruptive character to endosomes.

In a preferred embodiment, we describe a system for delivering apolynucleotide to a cell in vivo comprising: covalently linking atargeting group to a polynucleotide, covalently linking a secondtargeting group to a membrane active polymer, and injecting thepolynucleotide and membrane active polymer into an organism.

Further objects, features, and advantages of the invention will beapparent from the following detailed description when taken inconjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Reaction scheme for polymerization of polyvinylether polymers.

FIG. 2. Drawing illustrating several embodiments of masking agents ofthe invention.

FIG. 3. Diagram illustrating polynucleotide-polymer reversibleconjugates and reversible masking of the conjugate by maleamates.

FIG. 4. Micrograph showing targeted delivery to liver hepatocytes ofPBAVE polymer masked with A) CDM-PEG (left) or B) CDM-PEG plusCDM-Pip-LBA.

FIG. 5. Micrograph showing delivery of a masked polynucleotide-polymerconjugate to hepatocytes in vivo.

FIG. 6. Graph illustrating target gene knockdown in vivo followingpolyconjugate mediated delivery of ppara siRNA.

FIG. 7. Micrographs showing delivery of ds DNA to the liver in vivo with(A) DNA-DW1360-CDM-PEG/NAG, (B) DNA+DW1360-CDM-PEG/NAG (polynucleotidenot conjugated to the polymer, (C) DNA-DW1360-CDM-PEG/glucose, and (D)DNA-DW1360-CDM-PEG/Mannose. Cy3-labeled DNA=upper left quadrant in eachpanel, ALEXA®-488 phalloidin stained actin=upper right quadrant,nuclei=lower left quadrant, and composite picture=lower right quadrant.

FIG. 8. Graphs and blot illustrating knockdown of target gene expressionin livers of mice after intravenous injection of siRNA polyconjugates.(A) Reduction of apoB mRNA levels in liver after treatment with apoBsiRNA polyconjugates. (B) Serum levels of apoB-100 protein in apoB siRNApolyconjugate-treated mice. (C) Reduction of ppara mRNA levels in liverafter intravenous injection of ppara siRNA polyconjugates.

FIG. 9. Graph illustrating apoB-1 siRNA polyconjugate dose response. (A)Knockdown of apoB mRNA after injection of serial dilutions of the apoB-1siRNA polyconjugate. (B) Knockdown of apoB mRNA after injection ofvarying amounts of siRNA.

FIG. 10. Graph illustrating cholesterol levels in apoB-1 siRNApolyconjugate treated mice.

FIG. 11. Micrographs showing lipid accumulation in mouse liver followingpolyconjugate delivery of (A) ApoB siRNA or (B) GL3 negative controlsiRNA, or (C) saline alone.

FIG. 12. Graph illustrating inhibition of gene expression (A), andresultant decrease in serum cholesterol (B) and day 2-15 followingadministration of apoB-1 siRNA conjugate in mice on day 0.

FIG. 13. Micrograph showing in vivo delivery antibodies to hepatocytesvia administration of antibody reversibly conjugated to DW1360 andmasked with CDM-PEG and CDM-NAG. Upper left quadrant shows labeledantibodies, Upper right quadrant shows actin, lower left quadrant showsnuclei, and lower right quadrant shows a composite image.

FIG. 14. Graph illustrating gene knockdown in primary hepatocytestransfected with ApoB siRNA delivered with a commercial in vitrotransfection reagent or with varying amounts of ApoB siRNApolyconjugate.

FIG. 15. Graph illustrating transfection of siRNA-conjugates toHepa-1c1c7 cells in vitro: Conjugate alone=masked siRNA-PLL.

FIG. 16. Graph illustrating fluorescence of DPH in the presence orPBAVE.

FIG. 17. Graphs illustrating A) Elution profiles of PEG standards onShodex SB-803 column, B) Calibration plot for Shodex SB-803 column, andC) Molecular weight calculation for PBAVE polymer.

FIG. 18. Graphs illustrating A) Elution profiles of Cy3-labeled nucleicacid standards and masked polynucleotide-polymer conjugate, B)Calibration plot for the Sephacryl S-500 size exclusion chromatography,and C) Molecular weight calculation for the maskedpolynucleotide-polymer conjugate.

FIG. 19. Micrographs showing in vivo polynucleotide delivery to mousegalactose receptor positive tumors (A-B) or galactose receptor negativetumors (C-D) using oligonucleotide-polymer-CDM/PEG/NAG conjugates (A, C)or oligonucleotide-polymer-CDM/PEG/glucose conjugates (B, D).

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to compounds, compositions, andmethods useful for delivering polynucleotides or other cell-impermeablemolecules to mammalian cells. Described herein is a polyconjugate systemfor delivering polynucleotides or other membrane impermeable moleculesto cells in vivo. The polyconjugate system incorporates targeting,anti-opsonization, anti-aggregation, and transfection activities into asmall sub 50 nanometer delivery vehicle. A key component of the systemis the reversibility of bonds connecting the component parts.

A first component of the described in vivo polynucleotide deliveryconjugates comprises a physiologically reversible covalent linkage ofthe polynucleotide to a membrane active polymer. Covalent attachment ofthe polynucleotide to the membrane active polymer ensures that thepolynucleotide is not readily dissociated from the polymer whenadministered in vivo, or ex vivo in the presence of serum, and thusprovides for co-delivery of the polynucleotide with the membrane activepolymer to the target cell. Covalent attachment of a polynucleotide to apolymer can, however, render the polynucleotide inactive. Attachment ofthe polynucleotide to the membrane active polymer is thereforeaccomplished through a physiologically reversible linkage or bond. Byusing a physiologically reversible linkage, the polynucleotide can becleaved from the polymer, releasing the polynucleotide to engage infunctional interactions with cell components. By choosing an appropriatereversible linkage, it is possible to form a conjugate that releases thepolynucleotide only after it has been delivered to a desired location,such as a cell cytoplasm.

A second component of the described in vivo polynucleotide deliveryconjugates comprises a reversible modification of the membrane activepolymer. Reversible modification of the membrane active polymer reducesnon-productive serum and non-target cell interactions and reducestoxicity. The masking agent can also add a desired function to theconjugate such as enhancing target cell interaction or enhancingendocytosis of the conjugate. The polymer is masked by covalentattachment of a masking agent to the polymer via a physiologicallyreversible covalent linkage. The masking agent can be a stericstabilizer, targeting group, or charge modifier. The masking agents canshield the polymer from non-specific interactions, increase circulationtime, enhance specific interactions, inhibit toxicity, or alter thecharge of the polymer. Each of these modifications may alter themembrane activity of the polymer, rendering the modified polymer unableto facilitate delivery of the polynucleotide. Attachment of the maskingagent to the membrane active polymer is therefore accomplished through aphysiologically reversible bond. By using a physiologically reversiblelinkage, the masking agent can be cleaved from the polymer, therebyunmasking the polymer and restoring activity of the unmasked polymer. Bychoosing an appropriate reversible linkage, it is possible to form aconjugate that restores activity of the membrane active polymer after ithas been delivered or targeted to a desired cell type or cellularlocation.

The invention includes conjugate delivery systems of the generalstructure:N-L¹-P-(L²-M)_(y),wherein N is a polynucleotide or other cell impermeable molecule, L¹ isa reversible linkage, P is a polymer, L² is a second reversible linkage,and M is a masking agent. Masking agent M shields or modifies a propertyor interaction of P. y is an integer greater than 0. A preferred polymeris a membrane active polymer. Another preferred polymer is atransfection polymer. A plurality of masking agents may be linked to asingle polymer. The plurality of masking agents may be linked to thepolymer via a plurality of reversibly linkages. Upon cleavage ofreversible linkage L², the masked property or interaction is restored topolymer P. Masking agent M can add an activity, such as cell receptorbinding, to polymer P. The reversible bond of L¹ may be the same ordifferent than (orthogonal to) the reversible bond of L². The reversiblebond of reversible linkage L¹ or L² is chosen such that cleavage occursin a desired physiological condition, such as that present in a desiredtissue, organ, and sub-cellular location or in response to the additionof a pharmaceutically acceptable exogenous agent. Polynucleotide N andmasking agent M may be attached to polymer P anywhere along the lengthof polymer P.

Polymers

A polymer is a molecule built up by repetitive bonding together ofsmaller units called monomers. A polymer can be linear, branchednetwork, star, comb, or ladder type. The main chain of a polymer iscomposed of the atoms whose bonds are required for propagation ofpolymer length. For example, in poly-L-lysine, the carbonyl carbon,α-carbon, and α-amine groups are required for the length of the polymerand are therefore main chain atoms. A side chain of a polymer iscomposed of the atoms whose bonds are not required for propagation ofpolymer length.

A polymer can be a homopolymer in which a single monomer is used or apolymer can be copolymer in which two or more different monomers areused. Copolymers may by alternating, random (statistical), block andgraft (comb).

Alternating polymers contain monomers in a defined repeating order, suchas -[A-B]_(n)-. The monomers of alternating polymers typically do nothomopolymerize, instead reacting together to yield the alternatingcopolymer.

The monomers in random copolymers have no definite order or arrangementalong any given chain, such as: -A_(n)-B_(m)-. The general compositionsof such polymers are reflective of the ratio of input monomers. However,the exact ratio of one monomer to another may differ between chains. Thedistribution of monomers may also differ along the length of a singlepolymer. Also, the chemical properties of a monomer may affect its rateof incorporation into a random copolymer and its distribution within thepolymer. Thus, while the ratio of monomers in a random polymer isdependent on the input ratio of monomer, the input ratio may not matchexactly the ratio of incorporated monomers.

A block polymer has a segment or block of one polymer (polyA) followedby a segment or block of a second polymer (polyB): i.e., -polyA-polyB-.The result is that different polymer chains are joined in a head-to-tailconfiguration. Thus, a block polymer is a linear arrangement of blocksof different monomer composition. Generally, though not required, thepolymer blocks are homopolymers with polymer A being composed of adifferent monomer than polymer B. A diblock copolymer is polyA-polyB,and a triblock copolymer is polyA-polyB-polyA. If A is a hydrophilicgroup and B is hydrophobic group, the resultant block copolymer can beregarded a polymeric surfactant. A graft polymer is a type of blockcopolymer in which the chains of one monomer are grafted onto sidechains of the other monomer, as in (where n, m, x, y, and z areintegers):

Monomers

A wide variety of monomers can be used in polymerization processes.Monomers can be cationic, anionic, zwitterionic, hydrophilic,hydrophobic, lipophilic, amphipathic, or amphoteric. Monomers canthemselves be polymers. Monomers can contain chemical groups that can bemodified before or after polymerization. Such monomers may have reactivegroups selected from the group comprising: amine (primary, secondary,and tertiary), amide, carboxylic acid, ester, hydrazine, hydrazide,hydroxylamine, alkyl halide, aldehyde, and ketone. Preferably, thereactive group can be modified after conjugation of the polynucleotide,or in aqueous solution.

To those skilled in the art of polymerization, there are severalcategories of polymerization processes. For example, the polymerizationcan be chain or step.

Step Polymerization

In step polymerization, the polymerization occurs in a stepwise fashion.Polymer growth occurs by reaction between monomers, oligomers andpolymers. No initiator is needed since the same reaction occursthroughout and there is no termination step, so that the end groups arestill reactive. The polymerization rate decreases as the functionalgroups are consumed.

A polymer can be created using step polymerization by using monomersthat have two reactive groups (A and B) in the same monomer(heterobifunctional), wherein A comprises a reactive group and Bcomprises an A-reactive group (a reactive group which forms a covalentlybond with A). Polymerization of A-B yields -[A-B]_(n)-. Reactive groupsA and B can be joined by a covalent bond or a plurality of covalentbonds, thereby forming the polymer monomer. A polymer can also becreated using step polymerization by using homobifunctional monomerssuch that A-A+B-B yields -[A-A-B-B]_(n)-. Generally, these reactions caninvolve acylation or alkylation. The two reactive groups of a monomercan be joined by a single covalent bond or a plurality of covalentbonds.

If reactive group A is an amine then B is an amine-reactive group, whichcan be selected from the group comprising: isothiocyanate, isocyanate,acyl azide, N-hydroxy-succinimide, sulfonyl chloride, aldehyde(including formaldehyde and glutaraldehyde), ketone, epoxide, carbonate,imidoester, carboxylate activated with a carbodiimide, alkylphosphate,arylhalides (difluoro-dinitrobenzene), anhydride, acid halide,p-nitrophenyl ester, o-nitrophenyl ester, pentachlorophenyl ester,pentafluorophenyl ester, carbonyl imidazole, carbonyl pyridinium, andcarbonyl dimethylaminopyridinium. In other terms when reactive group Ais an amine then B can be acylating or alkylating agent or aminationagent.

If reactive group A is a sulfhydryl(thiol) then B is a thiol-reactivegroup, which can be select from the group comprising: iodoacetylderivative, maleimide, aziridine derivative, acryloyl derivative,fluorobenzene derivatives, and disulfide derivative (such as a pyridyldisulfide or 5-thio-2-nitrobenzoic acid (TNB) derivatives).

If reactive group A is carboxylate then reactive group B is acarboxylate-reactive group, which can be selected from the groupcomprising: diazoacetate and an amine in which a carbodiimide is used.Other additives may be utilized such as carbonyldiimidazole,dimethylamino pyridine (DMAP), N-hydroxysuccinimide or alcohol usingcarbodiimide and DMAP.

If reactive group A is a hydroxyl then reactive group B is ahydroxyl-reactive group, which can be selected from the groupcomprising: epoxide, oxirane, an activated carbamate, activated ester,and alkyl halide.

If reactive group A is an aldehyde or ketone then reactive group B is aaldehyde- or ketone-reactive group, which can be selected from the groupcomprising: hydrazine, hydrazide derivative, amine (to form a SchiffBase that may or may not be reduced by reducing agents such as NaCNBH₃),and hydroxyl compound.

A polymer can be created using step polymerization by using bifunctionalmonomers and another agent, such that that A-A plus another agent yields-[A-A]_(n)-.

If reactive group A is a sulfhydryl(thiol) group then it can beconverted to disulfide bonds by oxidizing agents such as iodine (I₂),sodium periodate (NaIO₄), or oxygen (O₂). If reactive group A can is anamine, it can be converted to a thiol by reaction with 2-Iminothiolate(Traut's reagent) which then undergoes oxidation and disulfideformation. Disulfide derivatives (such as a pyridyl disulfide or TNBderivatives) can also be used to catalyze disulfide bond formation.

Reactive groups A or B in any of the above examples can also be aphotoreactive group such as aryl azide (including halogenated arylazide), diazo, benzophenone, alkyne, or diazirine derivative.

Reactions of the amine, hydroxyl, sulfhydryl, or carboxylate groupsyield chemical bonds that are described as amides, amidines, disulfides,ethers, esters, enamines, imines, ureas, isothioureas, isoureas,sulfonamides, carbamates, alkylamine bonds (secondary amines), andcarbon-nitrogen single bonds in which the carbon contains a hydroxylgroup, thioether, diol, hydrazone, diazo, or sulfone.

Chain Polymerization

In chain-reaction polymerization growth of the polymer occurs bysuccessive addition of monomer units to a limited number of growingchains. The initiation and propagation mechanisms are different andthere is typically a chain-terminating step. Chain polymerizationreactions can be radical, anionic, or cationic. Monomers for chainpolymerization may be selected from the groups comprising: vinyl, vinylether, acrylate, methacrylate, acrylamide, and methacrylamide groups.Chain polymerization can also be accomplished by cycle or ring openingpolymerization. Several different types of free radical initiators canbe used including, but not limited to: peroxides, hydroxy peroxides, andazo compounds such as 2,2′-Azobis(-amidinopropane)dihydrochloride (AAP).

Transfection Activity

The term transfection refers to the transfer of a polynucleotide orother biologically active compound from outside a cell to inside a cellsuch that the polynucleotide or biologically active compound isfunctional. Examples of transfection reagents for delivery ofpolynucleotides to cells in vitro include, but are not limited to:liposomes, lipids, polyamines, calcium phosphate precipitates, histoneproteins, polyethylenimine, and polyampholyte complexes, andcombinations of these. Many in vitro transfection reagents are cationic,which to allows the reagent to associate with, or form a complex with,negatively charged nucleic acids via electrostatic interaction.

Membrane Active Polymers

Membrane active polymers are amphipathic polymers that are able toinduce one or more of the following effects upon a biological membrane:an alteration or disruption of the membrane that allows non-membranepermeable molecules to enter a cell or cross the membrane, poreformation in the membrane, fission of membranes, or disruption ordissolving of the membrane. As used herein, a membrane, or cellmembrane, comprises a lipid bilayer. Membrane active polymers of theinvention include those polymers that facilitate delivery of apolynucleotide or other membrane impermeable molecule from outside acell to inside the cell, and preferably to the cytoplasm of the cell.The alteration or disruption of the membrane can be functionally definedby the polymer's or compound's activity in at least one the followingassays: red blood cell lysis (hemolysis), liposome leakage, liposomefusion, cell fusion, cell lysis, and endosomal release. Membrane activepolymers that can cause lysis of cell membranes are also termed membranelytic polymers. Membrane active polymers that can cause disruption ofendosomes or lysosomes are considered endosomolytic. The effect ofmembrane active polymers on a cell membrane may be transient. Membraneactivity of a polymer is derived from its affinity for the membrane,which causes a denaturation or deformation of the bilayer structure.Membrane active polymers may be synthetic or non-natural amphipathicpolymers. Membrane active polymers may have in vitro transfectionactivity. Membrane active polymers may be cationic, anionic,amphipathic, amphoteric, surface active, or combinations of these.

Delivery of a polynucleotide, or other membrane impermeable molecule, toa cell is mediated by the membrane active polymer disrupting ordestabilizing the plasma membrane or an internal vesicle membrane (suchas an endosome or lysosome), including forming a pore in the membrane,or disrupting endosomal or lysosomal function.

As used herein, membrane active polymers are distinct from a class ofpolymers termed cell penetrating peptides or polymers represented bycompounds such as the arginine-rich peptide derived from the HIV TATprotein, the antennapedia peptide, VP22 peptide, transportan,arginine-rich artificial peptides, small guanidinium-rich artificialpolymers and the like. While cell penetrating compounds appear totransport some molecules across a membrane, from one side of a lipidbilayer to other side of the lipid bilayer, apparently without requiringendocytosis and without disturbing the integrity of the membrane, theirmechanism is not understood and the activity itself is disputed.

Endosomolytic Polymers

Endosomolytic polymers are polymers that, in response to a change in pH,are able to cause disruption or lysis of an endosome or provide forescape of a normally membrane-impermeable compound, such as apolynucleotide or protein, from a cellular internal membrane-enclosedvesicle, such as an endosome or lysosome. Endosomal release isimportance for the delivery of a wide variety of molecules which areendocytosed but incapable of diffusion across cellular membranes.Endosomolytic polymers undergo a shift in their physico-chemicalproperties over a physiologically relevant pH range (usually pH 5.5-8).This shift can be a change in the polymer's solubility, ability tointeract with other compounds, and a shift in hydrophobicity orhydrophilicity. Exemplary endosomolytic polymers can have pH-titratablegroups or pH-labile groups or bonds. As used herein, pH-titratablegroups reversibly accept or donate protons in water as a function of pHunder physiological conditions, i.e. a pH range of 4-8. pH-titratablegroups have pK_(a)'s in the range of 4-8 and act as buffers within thispH range. Thus, pH-titratable groups gain or lose charge in the lower pHenvironment of an endosome. Groups titratable at physiological pH can bedetermined experimentally by conducting an acid-base titration andexperimentally determining if the group buffers within the pH-range of4-8. Examples of groups that can exhibit buffering within this pH rangeinclude but are not limited to: carboxylic acids, imidazole,N-substituted imidazole, pyridine, phenols, and polyamines. Polymerswith pH-titratable groups may disrupt internal vesicles by the so-calledproton sponge effect. A reversibly masked membrane active polymer,wherein the masking agents are attached to the polymer via pH labilebonds, can therefore be considered to be an endosomolytic polymer.

A subset of endosomolytic compounds is fusogenic compounds, includingfusogenic peptides. Fusogenic peptides can facilitate endosomal releaseof agents such as oligomeric compounds to the cytoplasm. It is believedthat fusogenic peptides change conformation in acidic pH, effectivelydestabilizing the endosomal membrane thereby enhancing cytoplasmicdelivery of endosomal contents. Example fusogenic peptides includepeptides derived from polymyxin B, influenza HA2, GAL4, KALA, EALA,melittin and melittin-derived peptides, Alzheimer β-amyloid peptide, andthe like.

Polyampholytes

A polyampholyte is a polymer containing both anionic and cationicmonomer units. More specifically, as used herein, a polyampholytecontains a plurality of anionic monomers and a plurality of cationicunits. A polyampholyte polymer may optionally contain non-ionic monomerunits. In aqueous solutions polyampholytes precipitate near theirisoelectric points. A polyampholyte can be formed by polymerizinganionic and cationic monomers, including polymeric monomers.Alternatively, a polyampholyte can be formed by modifying more than one,but not all, of the charged groups on a polymer. If the charged groupsare reversibly modified, a reversible polyampholyte is formed.

Amphipathic

Amphipathic, or amphiphilic, polymers are well known and recognized inthe art and have both hydrophilic (polar, water-soluble) and hydrophobic(non-polar, lipophilic, water-insoluble) groups or parts. Hydrophilicgroups indicate in qualitative terms that the chemical moiety iswater-preferring. Typically, such chemical groups are water soluble, andare hydrogen bond donors or acceptors with water. A hydrophilic groupcan be charged or uncharged. Charged groups can be positively charged(anionic) or negatively charged (cationic) or both. An amphipathiccompound can be a polyanion, a polycation, a zwitterion, or apolyampholyte. Examples of hydrophilic groups include compounds with thefollowing chemical moieties; carbohydrates, polyoxyethylene, certainpeptides, oligonucleotides and groups containing amines, amides, alkoxyamides, carboxylic acids, sulfurs, or hydroxyls. Hydrophobic groupsindicate in qualitative terms that the chemical moiety iswater-avoiding. Typically, such chemical groups are not water soluble,and tend not to form hydrogen bonds. Lipophilic groups dissolve in fats,oils, lipids, and non-polar solvents and have little to no capacity toform hydrogen bonds. Hydrocarbons (containing 2 or more carbon atoms),certain substituted hydrocarbons, cholesterol, cholesterol derivatives,and certain peptides are examples of hydrophobic groups. As used herein,with respect to amphipathic polymers, a part is defined as a moleculederived when one covalent bond is broken and replaced by hydrogen. Forexample, in butyl amine, a breakage between the carbon and nitrogenbonds, with replacement with hydrogens, results in ammonia (hydrophilic)and butane (hydrophobic). However, if 1,4-diaminobutane is cleaved atnitrogen-carbon bonds, and replaced with hydrogens, the resultingmolecules are again ammonia (2×) and butane. However, 1,4,-diaminobutaneis not considered amphipathic because formation of the hydrophobic partrequires breakage of two bonds.

A naturally occurring polymer is a polymer that can be found in nature.Examples include polynucleotides, proteins, collagen, andpolysaccharides, (starches, cellulose, glycosaminoglycans, chitin, agar,agarose). A natural polymer can be isolated from a biologically sourceor it can be synthetic. A synthetic polymer is formulated ormanufactured by a chemical process “by man” and is not created by anaturally occurring biological process. A non-natural polymer is asynthetic polymer that is not made from naturally occurring (animal orplant) materials or monomers (such as: amino acids, nucleotides, andsaccharides). A polymer may be fully or partially natural, synthetic, ornon-natural.

A polymer may have one or more labile, or cleavable, bonds. If thelabile bonds are cleavable in physiological conditions or cellularphysiological conditions, the polymer is biodegradable. The cleavablebond may either be in the main-chain or in a side chain. If thecleavable bond occurs in the main chain, then cleavage of the bondresults in a decrease in polymer length and the formation of two or moremolecules. For example, smaller polyvinylether polymers can be joinedvia labile bonds to form large polyvinylether polymers. If the cleavablebond occurs in the side chain, then cleavage of the bond results in lossof side chain atoms from the polymer.

Disclosed herein is a class of amphipathic polyvinyl random copolymers.The polyvinyl copolymers may be synthesized from two, three, or moredifferent monomers. Preferred monomers may be selected from the listcomprising: charged vinyl ether, vinyl ether containing a protectedionic group, phthalimide-protected amine vinyl ether, acyl vinyl ether,alkyl vinyl ether, lower alkyl vinyl ether, higher alkyl vinyl ether,butyl vinyl ether, dodecyl vinyl ether, octadecyl vinyl ether,cholesterol vinyl ether, and other hydrocarbon-containing hydrophobicvinyl ethers.

A class of particularly useful polymers comprise: amine-containingpolyvinylether random copolymers polymerized with three monomers:phthalimide monomers, small hydrophobic monomers, and large hydrophobicmonomers. De-protection of the phthalimide monomer yields an aminemonomer. Small hydrophobic monomers comprise carbon-hydrogen chains withtwo to six carbon atoms. Large hydrophobic monomers comprisecarbon-hydrogen chains with about ten to about 22 carbon atoms, or about12 to about 18 carbon atoms. Medium hydrophobic monomers comprisecarbon-hydrogen chains with about 6 to about 10 carbon atoms. Preferredamino polyvinylether polymers comprise: amino/butyl/octadecylpolyvinylether or amino/butyl/dodecyl polyvinylether.

The biophysical properties of the poly(vinylether) random copolymers aredetermined by the particular monomers chosen, the ratio at which theyare incorporated into the polymer, and the size of the polymer.Different polymers can be made by altering the feed ratio of monomers inthe polymerization reaction. While the incorporated ratio of monomers ina polymer can be the same as the feed ratio of monomers, the ratios canbe different. Whether the monomers are incorporated at the feed ratio orat a different ratio, it is possible to alter the feed ratio of monomersto achieve a desired monomer incorporation ratio. A preferred aminopolyvinylether is a water soluble membrane active amine/butyl/octadecylor amine/butyl/dodecyl random tripolymer. It is possible to synthesizepolymers with similar amine and hydrophobic monomer content frompolyamines such as: polyethyleneimine, polylysine, polyvinylamine, andpolyallylamine, or other polymers such as polyvinylalcohol throughmodification of the side chains of these polymers.

Preferred membrane active polymers of the invention are water soluble at1 mg/ml or greater. Preferred membrane active polymers of the inventionare surface active. Membrane active polymers of the invention arepreferably in the size range of about 5 kDa to about 100 kDa, morepreferably about 7.5 kDa to about 50 kDa, and more preferably about 10kDa to about 30 kDa.

Masking Agent

Polymers capable of delivering a polynucleotide from outside a cell tothe cytoplasm of a cell are frequently toxic or have poorbio-distribution in vivo. Therefore, it is necessary to mask propertiesof the polymers that cause the toxicity or poor bio-distribution.Because modifying the polymer to mask these properties can alsoinactivate the transfection activity or membrane activity of thepolymer, masking agents are linked to the polymer via physiologicallyreversible linkages. Cleavage of the linkage restores the shielded ormasked property of the polymer.

As used herein, a masking agent comprises a molecule which, when linkedto a polymer, shields, inhibits or inactivates one or more properties(biophysical or biochemical characteristics) of the polymer. A maskingagent can also add an activity or function to the polymer that thepolymer did not have in the absence of the asking agent. Properties ofpolymers that may be masked include: membrane activity, endosomolyticactivity, charge, effective charge, transfection activity, seruminteraction, cell interaction, and toxicity. Masking agents can alsoinhibit or prevent aggregation of the polynucleotide-polymer conjugatein physiological conditions. Masking agents of the invention may beselected from the group consisting of: steric stabilizers, targetinggroups, and charge modifiers. Multiple masking agents can be reversiblylinked to a single polymer. To inactivate a property of a polymer, itmay be necessary to link more than one masking agent to the polymer. Asufficient number of masking agents are linked to the polymer to achievethe desired level of inactivation. The desired level of modification ofa polymer by attachment of masking agent(s) is readily determined usingappropriate polymer activity assays. For example, if the polymerpossesses membrane activity in a given assay, a sufficient level ofmasking agent is linked to the polymer to achieve the desired level ofinhibition of membrane activity in that assay. A sufficient number ofmasking agent can be reversibly linked to the polymer to inhibitaggregation of the polymer in physiologically conditions. More than onespecies of masking agent may be used. For example, both stericstabilizers and targeting groups may be linked to a polymer. Stericstabilizers and targeting groups may or may not also function as chargemodifiers. The masking agents of the invention are reversibly linked tothe polymer. As used herein, a masking agent is reversibly linked to apolymer if reversal of the linkage results in restoration of the maskedactivity of the polymer. Masking agents are linked to the polymerthrough the formation of reversible covalent linkages with reactivegroups on the polymer. Reactive groups may be selected from the groupscomprising: amines, alcohols, thiols, hydrazides, aldehydes, carboxyls,etc. From one to all of the reactive groups or charged groups on apolymer may be reversibly modified. In one embodiment, at least twomasking agents are reversibly linked to the polymer. In anotherembodiment, masking agents are reversibly linked to about 20%, 30%, 40%,50%, 60%, 70%, or 80% of the reactive groups on the polymer. In anotherembodiment, masking agents are reversibly linked to about 20%, 30%, 40%,50%, 60%, 70%, or 80% of the charged groups on the polymer. In anotherembodiment, the percentage of masking agents reversibly linked thepolymer to charged groups on the polymer is about 20%, 30%, 40%, 50%,60%, 70%, or 80%.

As used herein, a polymer is masked if one or more properties of thepolymer is inhibited or inactivated by attachment of one or more maskingagents. A polymer is reversibly masked if cleavage of bonds linking themasking agents to the polymer results in restoration of the polymer'smasked property.

Preferred masking agents of the invention are able to modify the polymer(form a reversible bond with the polymer) in aqueous solution. Ofparticular utility is a maleic anhydride derivative in which R¹ ismethyl (—CH₃) and R² is a propionic acid group (—(CH₂)₂CO₂H) oresters/amides derived from the acid.

As used herein, a steric stabilizer is a natural, synthetic, ornon-natural non-ionic hydrophilic polymer that prevents intramolecularor intermolecular interactions of polymer to which it is attachedrelative to the polymer containing no steric stabilizer. A stericstabilizer hinders a polymer from engaging in electrostaticinteractions. Electrostatic interactions are the non-covalentassociation of two or more substances due to attractive forces betweenpositive and negative charges. Steric stabilizers can inhibitinteraction with blood components and therefore opsonization,phagocytosis and uptake by the reticuloendothelial system. Stericstabilizers can thus increase circulation time of molecules to whichthey are attached. Steric stabilizers can also inhibit aggregation of apolymer. Preferred steric stabilizers are polyethylene glycol (PEG) andPEG derivatives. As used herein, a preferred PEG can have about 1-500ethylene glycol monomers, 2-20 ethylene glycol monomers, 5-15 ethyleneglycol monomers, or about 10 ethylene glycol monomers. As used herein, apreferred PEG can also have a molecular weight average of about85-20,000 Daltons (Da), about 200-1000 Da, about 200-750 Da, or about550 Da. Other suitable steric stabilizers may be selected from the groupcomprising: polysaccharides, dextran, cyclodextrin, poly(vinyl alcohol),polyvinylpyrrolidone, 2-hydroxypropyl methacrylate (HPMA), and watersoluble cellulose ether.

Targeting groups, or ligands, are used for targeting or delivery apolymer to target cells or tissues, or specific cells types. Targetinggroups enhance the association of molecules with a target cell. Thus,targeting groups can enhance the pharmacokinetic or biodistributionproperties of a conjugate to which they are attached to improve cellulardistribution and cellular uptake of the conjugate. One or more targetinggroups can be linked to the membrane active polymer either directly orvia a linkage with a spacer. Binding of a targeting group, such as aligand, to a cell or cell receptor may initiate endocytosis. Targetinggroups may be monovalent, divalent, trivalent, tetravalent, or havehigher valency. Targeting groups may be selected from the groupcomprising: compounds with affinity to cell surface molecule, cellreceptor ligands, and antibodies, antibody fragments, and antibodymimics with affinity to cell surface molecules. A preferred targetinggroup comprises a cell receptor ligand. A variety of ligands have beenused to target drugs and genes to cells and to specific cellularreceptors. Cell receptor ligands may be selected from the groupcomprising: carbohydrates, glycans, saccharides (including, but notlimited to: galactose, galactose derivatives, mannose, and mannosederivatives), vitamins, folate, biotin, aptamers, and peptides(including, but not limited to: RGD-containing peptides, insulin, EGF,and transferrin). Examples of targeting groups include those that targetthe asialoglycoprotein receptor by using asialoglycoproteins orgalactose residues. For example, liver hepatocytes contain ASGPReceptors. Therefore, galactose-containing targeting groups may be usedto target hepatocytes. Galactose containing targeting groups include,but are not limited to: galactose, N-acetylgalactosamine,oligosaccharides, and saccharide clusters (such as:Tyr-Glu-Glu-(aminohexyl GalNAc)₃, lysine-based galactose clusters, andcholane-based galactose clusters). Further suitable conjugates caninclude oligosaccharides that can bind to carbohydrate recognitiondomains (CRD) found on the asialoglycoprotein-receptor (ASGP-R). Exampleconjugate moieties containing oligosaccharides and/or carbohydratecomplexes are provided in U.S. Pat. No. 6,525,031

As used herein, a charge modifier is a group that alters the charge ofan ionic group on a polymer. The charge modifier can neutralize acharged group on a polymer or reverse the charge, from positive tonegative or negative to positive, of a polymer ion. Charge modificationof a polyion can reduce the charge of the polyion, form a polyion ofopposite charge, or form a polyampholyte. Charge modification can alsobe used to form a polymer with a desired net charge or zeta potential.Conjugates with near neutral net charge or zeta potential are preferredfor in vivo delivery of polynucleotides. A preferred charge modifierreversibly modifies a charged, or ionic, group. Reversible chargemodifiers may be selected from the group comprising: CDM, DM,CDM-thioester, CDM-masking agent, CDM-steric stabilizer, CDM-ligand,CDM-PEG, and CDM-galactose.

Zeta potential is a physical property which is exhibited by any particlein suspension and is closely related to surface charge. In aqueousmedia, the pH of the sample is one of the most important factors thataffects zeta potential. When charge is based uponprotonated/deprotonation of bases/acids, the charge is dependent on pH.Therefore, a zeta potential value must include the solution conditions,especially pH, to be meaningful. For typical particles, the magnitude ofthe zeta potential gives an indication of the potential stability of thecolloidal system. If all the particles in suspension have a largenegative or positive zeta potential then they will tend to repel eachother and there will be no tendency for the particles to come together.However, if the particles have low zeta potential values then there willbe no force to prevent the particles coming together and flocculating.The general dividing line between stable and unstable suspensions fortypical particles is generally taken at either +30 or −30 mV. Particleswith zeta potentials more positive than +30 mV or more negative than −30mV are normally considered stable. We show here, that CDM-maskedpolynucleotide-polymer conjugates can form small particles, <20 nm or<10 nm (as measured by dynamic light scattering, analyticultracentrifugation, or atomic force microscopy), that are stabledespite having a zeta potential between +30 and −30 mV at physiologicalsalt and pH 9.

Net charge, or zeta potential of the polynucleotide-polymer conjugatesof the invention, can be controlled by the attachment of masking agentsor charge modifiers. Polymer charge, especially positive charge, canresult in unwanted interactions with serum components or non-targetcells. By shielding charge with steric stabilizers or modification ofcharge, conjugates can be readily made with an apparent surface chargenear neutral. By modifying charged groups on the polymer, serum stableparticles can be made in which the zeta potential, measured at pH 9, isbetween +30 and −30 mV, between +20 and −20 mV, between +10 and −10 mV,or between +5 and −5 mV. At pH 7, the net charge of the conjugate wouldbe expected to be more positive than at pH 9. Net charge, or surfacecharge, is a significant factor in delivery of polynucleotide complexesin vivo.

Labile Linkage

A linkage or linker is a connection between two atoms that links onechemical group or segment of interest to another chemical group orsegment of interest via one or more covalent bonds. For example, alinkage can connect a polynucleotide or masking agent to a polymer.Formation of a linkage may connect two separate molecules into a singlemolecule or it may connect two atoms in the same molecule. The linkagemay be charge neutral, or may bear a positive or negative charge. Areversible or labile linkage contains a reversible or labile bond. Alinkage may optionally include a spacer that increases the distancebetween the two joined atoms. A spacer may further add flexibilityand/or length to the linkage. Spacers may include, but are not belimited to: alkyl groups, alkenyl groups, alkynyl groups, aryl groups,aralkyl groups, aralkenyl groups, aralkynyl groups, each of which cancontain one or more heteroatoms, and heterocycles. Spacer groups arewell known in the art and the preceding list is not meant to limit thescope of the invention.

A reversible or labile bond is a covalent bond other than a covalentbond to a hydrogen atom that is capable of being selectively broken orcleaved under conditions that will not break or cleave other covalentbonds in the same molecule. More specifically, a reversible or labilebond is a covalent bond that is less stable (thermodynamically) or morerapidly broken (kinetically) under appropriate conditions than othernon-labile covalent bonds in the same molecule. Cleavage of a labilebond within a molecule may result in the formation of two or moremolecules. For those skilled in the art, cleavage or lability of a bondis generally discussed in terms of half-life (t_(1/2)) of bond cleavage,or the time required for half of the bonds to cleave. Orthogonal bondsare bonds that cleave under conditions that cleave one and not theother. Two bonds are considered orthogonal if their half-lives ofcleavage in a defined environment are 10-fold or more different from oneanother. Thus, reversible or labile bonds encompass bonds that can beselectively cleaved more rapidly than other bonds a molecule.

The presence of electron donating or withdrawing groups can be locatedin a molecule sufficiently near the cleavable bond such that theelectronic effects of the electron donating or withdrawing groupsinfluence the rate of bond cleavage. Electron withdrawing groups (EWG)are atoms or parts of molecules that withdraw electron density fromanother atom, bond, or part of the molecule wherein there is a decreasein electron density to the bond of interest (donor). Electron donatinggroups (EDG) are atoms or parts of molecules that donate electrons toanother atom, bond, or part of the molecule wherein there is anincreased electron density to the bond of interest (acceptor). Theelectron withdrawing/donating groups need to be in close enoughproximity to effect influence, which is typically within about 3 bondsof the bond being broken.

Another strategy for increasing the rate of bond cleavage is toincorporate functional groups into the same molecule as the labile bond.The proximity of functional groups to one another within a molecule canbe such that intramolecular reaction is favored relative to anintermolecular reaction. The proximity of functional groups to oneanother within the molecule can in effect result in locally higherconcentrations of the functional groups. In general, intramolecularreactions are much more rapid than intermolecular reactions. Reactivegroups separated by 5 and 6 atoms can form particularly labile bonds dueto the formation of 5 and 6-membered ring transition states. Examplesinclude having carboxylic acid derivatives (acids, esters, amides) andalcohols, thiols, carboxylic acids or amines in the same moleculereacting together to make esters, carboxylic and carbonate esters,phosphate esters, thiol esters, acid anhydrides or amides. Stericinteractions can also change the cleavage rate for a bond.

Appropriate conditions are determined by the type of labile bond and arewell known in organic chemistry. A labile bond can be sensitive to pH,oxidative or reductive conditions or agents, temperature, saltconcentration, the presence of an enzyme, or the presence of an addedagent. For example, certain peptide, ester, or saccharide linkers may becleaved in the presence of the appropriate enzyme. In another example,increased or decreased pH may be the appropriate conditions for apH-labile bond. In yet another example, oxidative conditions may be theappropriated conditions for an oxidatively labile bond. In yet anotherexample, reductive conditions may be the appropriate conditions for areductively labile bond. For instance, a disulfide constructed from twoalkyl thiols is capable of being broken by reduction in the presence ofthiols, without cleavage of carbon-carbon bonds. In this example, thecarbon-carbon bonds are non-labile to the reducing conditions.

The rate at which a labile group will undergo transformation can becontrolled by altering the chemical constituents of the moleculecontaining the labile group. For example, addition of particularchemical moieties (e.g., electron acceptors or donors) near the labilegroup can affect the particular conditions (e.g., pH) under whichchemical transformation will occur.

Molecules can contain one or more reversible or labile bonds. If morethan one reversible or labile bond is present in the molecule, and allof the reversible or labile bonds are of the same type (cleaved at aboutthe same rate under the same conditions), then the molecule containsmultiple reversible or labile bonds. If more than one reversible orlabile bond is present in the molecule, and the reversible or labilebonds are of different types (based either on the appropriate conditionsfor lability, or the chemical functional group of the labile bonds),then the molecule contains orthogonal reversible or labile bonds.Orthogonal reversible or labile bonds provides for the ability to cleaveone type of reversible or labile bond while leaving a second type ofreversible bond not cleaved or broken. In other words, two labile bondsare orthogonal to each other if one can be cleaved under conditions thatleave the other intact. Orthogonal protecting groups are well known inorganic chemistry and comprise orthogonal bonds.

As used herein, a physiologically labile bond is a labile bond that iscleavable under conditions normally encountered or analogous to thoseencountered within a mammalian body. Physiologically labile linkagegroups are selected such that they undergo a chemical transformation(e.g., cleavage) when present in certain physiological conditions.

As used herein, a cellular physiologically labile bond is a labile bondthat is cleavable under mammalian intracellular conditions. Mammalianintracellular conditions include chemical conditions such as pH,temperature, oxidative or reductive conditions or agents, and saltconcentration found in or analogous to those encountered in mammaliancells. Mammalian intracellular conditions also include the presence ofenzymatic activity normally present in a mammalian cell such as fromproteolytic or hydrolytic enzymes. A cellular physiologically labilebond may also be cleaved in response to administration of apharmaceutically acceptable exogenous agent. Physiologically labilebonds that are cleaved with a half life of less than 45 min. areconsidered very labile. Physiologically labile bonds that are cleavedwith a half life of less than 15 min are considered extremely labile.

Chemical transformation (cleavage of the labile bond) may be initiatedby the addition of a pharmaceutically acceptable agent to the cell ormay occur spontaneously when a molecule containing the labile bondreaches an appropriate intra- and/or extra-cellular environment. Forexample, a pH labile bond may be cleaved when the molecule enters anacidified endosome. Thus, a pH labile bond may be considered to be anendosomal cleavable bond. Enzyme cleavable bonds may be cleaved whenexposed to enzymes such as those present in an endosome or lysosome orin the cytoplasm. A disulfide bond may be cleaved when the moleculeenters the more reducing environment of the cell cytoplasm. Thus, adisulfide may be considered to be a cytoplasmic cleavable bond.

pH-labile bond: As used herein, a pH-labile bonds is a labile bond thatis selectively broken under acidic conditions (pH<7). Such bonds mayalso be termed endosomally labile bonds, since cell endosomes andlysosomes have a pH less than 7. The term pH-labile includes bonds thatare pH-labile, very pH-labile, and extremely pH-labile. pH labile bondsmay be selected from the group comprising:

-   -   a) ketals that are labile in acidic environments (pH less than        7, greater than 4) to form a diol and a ketone,    -   b) acetals that are labile in acidic environments (pH less than        7, greater than 4) to form a diol and an aldehyde,    -   c) imines or iminiums that are labile in acidic environments (pH        less than 7, greater than 4) to form an amine and an aldehyde or        a ketone,    -   d) silicon-oxygen-carbon linkages that are labile under acidic        conditions,    -   e) silicon-nitrogen (silazanes) linkages, and    -   f) silicon-carbon linkages (arylsilanes, vinylsilanes, and        allylsilanes).    -   g) maleamates-amide bonds synthesized from maleic anhydride        derivatives and amines    -   h) ortho esters    -   i) hydrazones    -   j) activated carboxylic acid derivatives (esters, amides)        designed to undergo acid catalyzed hydrolysis.    -   k) vinyl ethers

Organosilanes have long been utilized as oxygen protecting groups inorganic synthesis due to both the ease in preparation (of thesilicon-oxygen-carbon linkage) and the facile removal of the protectinggroup under acidic conditions. For example, silyl ethers andsilylenolethers, both posses such a linkage. Silicon-oxygen-carbonlinkages are susceptible to hydrolysis under acidic conditions formingsilanols and an alcohol (or enol). The substitution on both the siliconatom and the alcohol carbon can affect the rate of hydrolysis due tosteric and electronic effects. This allows for the possibility of tuningthe rate of hydrolysis of the silicon-oxygen-carbon linkage by changingthe substitution on either the organosilane, the alcohol, or both theorganosilane and alcohol to facilitate the desired affect. In addition,charged or reactive groups, such as amines or carboxylate, may be linkedto the silicon atom, which confers the labile compound with chargeand/or reactivity.

Hydrolysis of a silazane leads to the formation of a silanol and anamine. Silazanes are inherently more susceptible to hydrolysis than isthe silicon-oxygen-carbon linkage, however, the rate of hydrolysis isincreased under acidic conditions. The substitution on both the siliconatom and the amine can affect the rate of hydrolysis due to steric andelectronic effects. This allows for the possibility of tuning the rateof hydrolysis of the silizane by changing the substitution on either thesilicon or the amine to facilitate the desired affect.

Another example of a pH labile bond is the use of the acid labile enolether bond. The rate at which this labile bond is cleaved depends on thestructures of the carbonyl compound formed and the alcohol released. Forexample analogs of ethyl isopropenyl ether, which may be synthesizedfrom β-haloethers, have half-lives of roughly 2 min at pH 5. Analogs ofethyl cyclohexenyl ether, which may be synthesized from phenol ethers,have half-lives of roughly 14 min at pH 5.

Reaction of an anhydride with an amine forms an amide and an acid.Typically, the reverse reaction (formation of an anhydride and amine) isvery slow and energetically unfavorable. However, if the anhydride is acyclic anhydride, reaction with an amine yields a molecule in which theamide and the acid are in the same molecule, an amide acid. The presenceof both reactive groups (the amide and the carboxylic acid) in the samemolecule accelerates the reverse reaction. In particular, the product ofprimary amines with maleic anhydride and maleic anhydride derivatives,maleamic acids, revert back to amine and anhydride 1×10⁹ to 1×10¹³ timesfaster than its noncyclic analogues (Kirby 1980).

Reaction of an Amine with an Anhydride to Form an Amide and an Acid

Reaction of an Amine with a Cyclic Anhydride to Form an Amide Acid

Cleavage of the amide acid to form an amine and an anhydride ispH-dependent, and is greatly accelerated at acidic pH. This pH-dependentreactivity can be exploited to form reversible pH-sensitive bonds andlinkers. Cis-aconitic acid has been used as such a pH-sensitive linkermolecule. The γ-carboxylate is first coupled to a molecule. In a secondstep, either the α or β carboxylate is coupled to a second molecule toform a pH-sensitive coupling of the two molecules. The half life forcleavage of this linker at pH 5 is between 8 and 24 h.

Structures of Cis-Aconitic Anhydride and Maleic Anhydride

The pH at which cleavage occurs is controlled by the addition ofchemical constituents to the labile moiety. The rate of conversion ofmaleamic acids to amines and maleic anhydrides is strongly dependent onsubstitution (R² and R³) of the maleic anhydride system. When R² ismethyl, the rate of conversion is 50-fold higher than when R² and R³ arehydrogen. When there are alkyl substitutions at both R² and R³ (e.g.,2,3-dimethylmaleicanhydride) the rate increase is dramatic, 10,000-foldfaster than non-substituted maleic anhydride. The maleamate bond formedfrom the modification of an amine with 2,3-dimethylmaleic anhydride iscleaved to restore the anhydride and amine with a half-life between 4and 10 min at pH 5. It is anticipated that if R² and R³ are groupslarger than hydrogen, the rate of amide-acid conversion to amine andanhydride will be faster than if R² and/or R³ are hydrogen.

Very pH-labile bond: A very pH-labile bond has a half-life for cleavageat pH 5 of less than 45 min. The construction of very pH-labile bonds iswell-known in the chemical art.

Extremely pH-labile bonds: An extremely pH-labile bond has a half-lifefor cleavage at pH 5 of less than 15 min. The construction of extremelypH-labile bonds is well-known in the chemical art.

Linkage of Polynucleotide to Polymer

Most previous methods for delivery of polynucleotide to cells haverelied upon complexation of the anionic nucleic acid with a cationicdelivery agent such as a cationic polymer or cationic lipid.Electrostatic complexes with larger polynucleotides, e.g. plasmids, tendto aggregate and become large, >500 nm, in physiological solutions.Smaller polynucleotides, oligonucleotides, because of their small size,form unstable electrostatic complexes with potential delivery agents. Amore effective method for packaging polynucleotides is to covalentlylink the polynucleotide to the delivery vehicle. However, attachment tothe polymer can inhibit the activity of the polynucleotide in the cell.We have found that by attaching the polynucleotide to the polymer via areversible linker that is broken after the polynucleotide is deliveredto the cell, it is possible to delivery a functionally activepolynucleotide to a cell in vivo. The reversible linker is selected suchthat it undergoes a chemical transformation (e.g., cleavage) whenpresent in certain physiological conditions, (e.g., the reducingenvironment of the cell cytoplasm). Attachment of a polynucleotide to adelivery or membrane active polymer enhances delivery of thepolynucleotide to a cell in vivo. Release of the polynucleotide from thepolymer, by cleavage of the reversible bond, facilitates interaction ofthe polynucleotide with the appropriate cellular components foractivity.

In addition to polynucleotides, delivery of other membrane impermeablemolecules or molecules with low membrane permeability can be deliveredto cells in vivo using the described polyconjugate delivery vehicles.Molecules suitable for reversible attachment to a membrane activepolymer include: proteins, antibodies, antibody fragments, transcriptionfactors, small molecule drugs, anticancer drugs, and other syntheticmolecules including those that affect transcription.

Attachment of the membrane impermeable molecule to a membrane activepolymer enables the formation of an appropriately sized in vivo deliveryvehicle. The described molecular conjugates are essentially polymericand have similar size and targeting propertied as that observed forother polymers. For intravascular (system) delivery the vehicle needs tobe able to cross the endothelial barrier in order reach parenchymalcells of interest. The largest endothelia fenestrae (holes in theendothelial barrier) occur in the liver and have an average diameter ofabout 100 nm. The trans-epithelial pores in other organs are muchsmaller. For example, muscle endothelium can be described as a structurewhich has a large number of small pores with a radius of 4 nm, and a lownumber of large pores with a radius of 20-30 nm. The size of thepolynucleotide delivery vehicle is also important for the cellularuptake process. Cellular internalization through endocytosis is thoughtto be limited to complexes of about 100 nm in diameter, the size ofendocytic vesicles. The described conjugates are less than 100 nm, morepreferably less than 50 nM, and more preferably less than 20 nM or lessthan 10 nm.

Renal ultrafiltration is one of the main routes of elimination ofhydrophilic proteins, polymers and polymer-protein conjugates fromblood. Among the parameters affecting this process are chemicalcomposition, size, charge. Globular proteins larger than about 70 kDaare largely excluded from clearance by renal ultrafiltration.Conjugation of steric stabilizers such as PEG, therefore, decreasesrenal clearance by increasing the effective molecular size of polymersto which they are attached. Ultrafiltration of PEGs with molecularweight lower than 8 kDa is not restricted, while ultrafiltration of PEGsin the range of 8-30 kDa is governed by the molecular size. Withmolecular weight exceeding 30 kDa, PEG elimination is dramaticallydecreased. For this reason, masked polymer-polynucleotide conjugates inwhich the overall size is larger than about 10 kDa, larger than about 20kDa, or larger than about 30 kDa are preferred. In addition todecreasing kidney ultrafiltration, an increase molecule weight of apolymer can promote accumulation into permeable tissues, such as tumors,by the passive enhanced permeation and retention mechanism.

Polynucleotide

The term polynucleotide, or nucleic acid or polynucleic acid, is a termof art that refers to a polymer containing at least two nucleotides.Nucleotides are the monomeric units of polynucleotide polymers.Polynucleotides with less than 120 monomeric units are often calledoligonucleotides. Natural nucleic acids have a deoxyribose- orribose-phosphate backbone. A non-natural or synthetic polynucleotide isa polynucleotide that is polymerized in vitro or in a cell free systemand contains the same or similar bases but may contain a backbone of atype other than the natural ribose or deoxyribose-phosphate backbone.Polynucleotides can be synthesized using any known technique in the art.Polynucleotide backbones known in the art include: PNAs (peptide nucleicacids), phosphorothioates, phosphorodiamidates, morpholinos, and othervariants of the phosphate backbone of native nucleic acids. Basesinclude purines and pyrimidines, which further include the naturalcompounds adenine, thymine, guanine, cytosine, uracil, inosine, andnatural analogs. Synthetic derivatives of purines and pyrimidinesinclude, but are not limited to, modifications which place new reactivegroups on the nucleotide such as, but not limited to, amines, alcohols,thiols, carboxylates, and alkylhalides. The term base encompasses any ofthe known base analogs of DNA and RNA. The term polynucleotide includesdeoxyribonucleic acid (DNA) and ribonucleic acid (RNA) and combinationsof DNA, RNA and other natural and synthetic nucleotides.

DNA may be in form of cDNA, in vitro polymerized DNA, plasmid DNA, partsof a plasmid DNA, genetic material derived from a virus, linear DNA,vectors (P1, PAC, BAC, YAC, and artificial chromosomes), expressionvectors, expression cassettes, chimeric sequences, recombinant DNA,chromosomal DNA, an oligonucleotide, anti-sense DNA, or derivatives ofthese groups. RNA may be in the form of messenger RNA (mRNA), in vitropolymerized RNA, recombinant RNA, oligonucleotide RNA, transfer RNA(tRNA), small nuclear RNA (snRNA), ribosomal RNA (rRNA), chimericsequences, anti-sense RNA, interfering RNA, small interfering RNA(siRNA), microRNA (miRNA), ribozymes, external guide sequences, smallnon-messenger RNAs (snmRNA), untranslatedRNA (utRNA), snoRNAs (24-mers,modified snmRNA that act by an anti-sense mechanism), tiny non-codingRNAs (tncRNAs), small hairpin RNA (shRNA), or derivatives of thesegroups. In addition, DNA and RNA may be single, double, triple, orquadruple stranded. Double, triple, and quadruple strandedpolynucleotide may contain both RNA and DNA or other combinations ofnatural and/or synthetic nucleic acids.

A blocking polynucleotide is a polynucleotide that interferes with thefunction or expression of DNA or RNA. Blocking polynucleotides are nottranslated into protein but their presence or expression in a cellalters the expression or function of cellular genes or RNA. Blockingpolynucleotides cause the degradation of or inhibit the function ortranslation of a specific cellular RNA, usually an mRNA, in asequence-specific manner. Inhibition of an RNA can thus effectivelyinhibit expression of a gene from which the RNA is transcribed. As usedherein, a blocking polynucleotide may be selected from the listcomprising: anti-sense oligonucleotide, RNA interference polynucleotide,dsRNA, siRNA, miRNA, hRNA, ribozyme, hammerhead ribozyme, external guidesequence (U.S. Pat. No. 5,962,426), snoRNA, triple-helix formingoligonucleotide RNA Polymerase II transcribed DNA encoding a blockingpolynucleotide, RNA Polymerase III transcribed DNAs encoding a blockingpolynucleotide. Blocking polynucleotide can be DNA, RNA, combination ofRNA and DNA, or may contain non-natural or synthetic nucleotides.

Blocking polynucleotides may be polymerized in vitro, they may berecombinant, contain chimeric sequences, or derivatives of these groups.A blocking polynucleotide may contain ribonucleotides,deoxyribonucleotides, synthetic nucleotides, or any suitable combinationsuch that the target RNA or gene is inhibited.

An RNA interference (RNAi) polynucleotide is a molecule capable inducingRNA interference through interaction with the RNA interference pathwaymachinery of mammalian cells to degrade or inhibit translation ofmessenger RNA (mRNA) transcripts of a transgene in a sequence specificmanner. Two primary RNAi polynucleotides are small (or short)interfering RNAs (siRNAs) and micro RNAs (miRNAs). However, otherpolynucleotides have been shown to mediate RNA interference. RNAipolynucleotides may be selected from the group comprising: siRNA,microRNA, double-strand RNA (dsRNA), short hairpin RNA (shRNA), andexpression cassettes encoding RNA capable if inducing RNA interference.SiRNA comprises a double stranded structure typically containing 15-50base pairs and preferably 21-25 base pairs and having a nucleotidesequence identical (perfectly complementary) or nearly identical(partially complementary) to a coding sequence in an expressed targetgene or RNA within the cell. An siRNA may have dinucleotide 3′overhangs. An siRNA may be composed of two annealed polynucleotides or asingle polynucleotide that forms a hairpin structure. An siRNA moleculeof the invention comprises a sense region and an antisense region. Inone embodiment, the siRNA of the conjugate is assembled from twooligonucleotide fragments, wherein one fragment comprises the nucleotidesequence of the antisense strand of the siRNA molecule and a secondfragment comprises nucleotide sequence of the sense region of the siRNAmolecule. In another embodiment, the sense strand is connected to theantisense strand via a linker molecule, such as a polynucleotide linkeror a non-nucleotide linker. MicroRNAs (miRNAs) are small noncoding RNAgene products about 22 nt long that direct destruction or translationalrepression of their mRNA targets. If the complementarity between themiRNA and the target mRNA is partial, then translation of the targetmRNA is repressed, whereas if complementarity is extensive, the targetmRNA is cleaved. For miRNAs, the complex binds to target sites usuallylocated in the 3′ UTR of mRNAs that typically share only partialhomology with the miRNA. A “seed region”—a stretch of about 7consecutive nucleotides on the 5′ end of the miRNA that forms perfectbase pairing with its target—plays a key role in miRNA specificity.Binding of the RISC/miRNA complex to the mRNA can lead to either therepression of protein translation or cleavage and degradation of themRNA. Recent data indicate that mRNA cleavage happens preferentially ifthere is perfect homology along the whole length of the miRNA and itstarget instead of showing perfect base-pairing only in the seed region(Pillai et al. 2007).

Antisense oligonucleotide comprise a polynucleotide containing sequencethat is complimentary to a sequence present in a target mRNA. Theantisense oligonucleotide binds to (base pairs with) mRNA in a sequencespecific manner. This binding can prevent other cellular enzymes frombinding to the mRNA, thereby leading to inhibition of translation of themRNA or degradation of the mRNA.

External guide sequences are short antisense oligoribonucleotides thatinduce RNase P-mediated cleavage of a target RNA by forming a precursortRNA-like complex (U.S. Pat. No. 5,624,824).

Ribozymes are typically RNA oligonucleotides that contain sequencecomplementary to the target messenger RNA and an RNA sequence that actsas an enzyme to cleave the messenger RNA. Cleavage of the mRNA preventstranslation.

An oligonucleotide that forms the blocking polynucleotide can include aterminal cap moiety at the 5′-end, the 3′-end, or both of the 5′ and 3′ends. The cap moiety can be, but is not limited to, an inverted deoxyabasic moiety, an inverted deoxy thymidine moiety, a thymidine moiety,or 3′ glyceryl modification.

RNA polymerase II and III transcribed DNAs can be transcribed in thecell to produce small hairpin RNAs that can function as siRNA, separatesense and anti-sense strand linear siRNAs, ribozymes, or linear RNAsthat can function as antisense RNA. RNA polymerase III transcribed DNAscontain promoters selected from the list comprising: U6 promoters, IIIpromoters, and tRNA promoters. RNA polymerase II promoters include U1,U2, U4, and U5 promoters, snRNA promoters, microRNA promoters, and mRNApromoters.

Lists of known miRNA sequences can be found in databases maintained byresearch organizations such as Wellcome Trust Sanger Institute, PennCenter for Bioinformatics, Memorial Sloan Kettering Cancer Center, andEuropean Molecule Biology Laboratory, among others. Known effectivesiRNA sequences and cognate binding sites are also well represented inthe relevant literature. RNAi molecules are readily designed andproduced by technologies known in the art. In addition, there arecomputational tools that increase the chance of finding effective andspecific sequence motifs (Pei et al. 2006, Reynolds et al. 2004,Khvorova et al. 2003, Schwarz et al. 2003, Ui-Tei et al. 2004, Heale etal. 2005, Chalk et al. 2004, Amarzguioui et al. 2004).

The polynucleotides of the invention can be chemically modified. The useof chemically modified polynucleotide can improve various properties ofthe polynucleotide including, but not limited to: resistance to nucleasedegradation in vivo, cellular uptake, activity, and sequence-specifichybridization. Non-limiting examples of such chemical modificationsinclude: phosphorothioate internucleotide linkages, 2′-O-methylribonucleotides, 2′-deoxy-2′-fluoro ribonucleotides, 2′-deoxyribonucleotides, “universal base” nucleotides, 5-C-methyl nucleotides,and inverted deoxyabasic residue incorporation. These chemicalmodifications, when used in various polynucleotide constructs, are shownto preserve polynucleotide activity in cells while at the same time,dramatically increasing the serum stability of these compounds.Chemically modified siRNA can also minimize the possibility ofactivating interferon activity in humans.

In one embodiment, the chemically-modified RNAi polynucleotide of theinvention comprises a duplex having two strands, one or both of whichcan be chemically-modified, wherein each strand is about 19 to about 29(e.g., about 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29) nucleotides.In one embodiment, an RNAi polynucleotide of the invention comprises oneor more modified nucleotides while maintaining the ability to mediateRNAi inside a cell or reconstituted in vitro system. An RNAipolynucleotide can be modified wherein the chemical modificationcomprises one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, ormore) of the nucleotides. An RNAi polynucleotide of the invention cancomprise modified nucleotides as a percentage of the total number ofnucleotides present in the RNAi polynucleotide. As such, an RNAipolynucleotide of the invention can generally comprise modifiednucleotides from about 5 to about 100% of the nucleotide positions(e.g., 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95% or 100% of the nucleotide positions). Theactual percentage of modified nucleotides present in a given RNAipolynucleotide depends on the total number of nucleotides present in theRNAi polynucleotide. If the RNAi polynucleotide is single stranded, thepercent modification can be based upon the total number of nucleotidespresent in the single stranded RNAi polynucleotide. Likewise, if theRNAi polynucleotide is double stranded, the percent modification can bebased upon the total number of nucleotides present in the sense strand,antisense strand, or both the sense and antisense strands. In addition,the actual percentage of modified nucleotides present in a given RNAipolynucleotide can also depend on the total number of purine andpyrimidine nucleotides present in the RNAi polynucleotide. For example,wherein all pyrimidine nucleotides and/or all purine nucleotides presentin the RNAi polynucleotide are modified.

An RNAi polynucleotide modulates expression of RNA encoded by a gene.Because multiple genes can share some degree of sequence homology witheach other, an RNAi polynucleotide can be designed to target a class ofgenes with sufficient sequence homology. Thus an RNAi polynucleotide cancontain sequence that has complementarity to sequences that are sharedamongst different gene targets or are unique for a specific gene target.Therefore the RNAi polynucleotide can be designed to target conservedregions of a RNA sequence having homology between several genes therebytargeting several genes in a gene families (e.g., different geneisoforms, splice variants, mutant genes etc.). In another embodiment,the RNAi polynucleotide can be designed to target a sequence that isunique to a specific RNA sequence of a single gene.

The term complementarity refers to the ability of a polynucleotide toform hydrogen bond(s) with another polynucleotide sequence by eithertraditional Watson-Crick or other non-traditional types. In reference tothe polynucleotide molecules of the present invention, the binding freeenergy for a polynucleotide molecule with its target (effector bindingsite) or complementary sequence is sufficient to allow the relevantfunction of the polynucleotide to proceed, e.g., enzymatic mRNA cleavageor translation inhibition. Determination of binding free energies fornucleic acid molecules is well known in the art (Frier et al. 1986,Turner et al. 1987). A percent complementarity indicates the percentageof bases, in a contiguous strand, in a first polynucleotide moleculewhich can form hydrogen bonds (e.g., Watson-Crick base pairing) with asecond polynucleotide sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being50%, 60%, 70%, 80%, 90%, and 100% complementary). Perfectlycomplementary means that all the bases, in a contiguous strand of apolynucleotide sequence will hydrogen bond with the same number ofcontiguous bases in a second polynucleotide sequence.

By inhibit, down-regulate, or knockdown gene expression, it is meantthat the expression of the gene, as measured by the level of RNAtranscribed from the gene, or the level of polypeptide, protein orprotein subunit translated from the RNA, is reduced below that observedin the absence of the blocking polynucleotide-conjugates of theinvention. Inhibition, down-regulation, or knockdown of gene expression,with a polynucleotide delivered by the compositions of the invention, ispreferably below that level observed in the presence of a controlinactive nucleic acid, a nucleic acid with scrambled sequence or withinactivating mismatches, or in absence of conjugation of thepolynucleotide to the masked polymer.

A delivered polynucleotide can stay within the cytoplasm or nucleusapart from the endogenous genetic material. Alternatively, DNA canrecombine with (become a part of) the endogenous genetic material.Recombination can cause DNA to be inserted into chromosomal DNA byeither homologous or non-homologous recombination.

A polynucleotide can be delivered to a cell to express an exogenousnucleotide sequence, to inhibit, eliminate, augment, or alter expressionof an endogenous nucleotide sequence, or to affect a specificphysiological characteristic not naturally associated with the cell.

Polynucleotides may contain an expression cassette coded to express awhole or partial protein, or RNA. An expression cassette refers to anatural or recombinantly produced polynucleotide that is capable ofexpressing a sequence. The term recombinant as used herein refers to apolynucleotide molecule that is comprised of segments of polynucleotidejoined together by means of molecular biological techniques. Thecassette contains the coding region of the gene of interest along withany other sequences that affect expression of the sequence of interest.An expression cassette typically includes a promoter (allowingtranscription initiation), and a transcribed sequence. Optionally, theexpression cassette may include, but is not limited to: transcriptionalenhancers, non-coding sequences, splicing signals, transcriptiontermination signals, and polyadenylation signals. An RNA expressioncassette typically includes a translation initiation codon (allowingtranslation initiation), and a sequence encoding one or more proteins.Optionally, the expression cassette may include, but is not limited to,translation termination signals, a polyadenosine sequence, internalribosome entry sites (IRES), and non-coding sequences.

The polynucleotide may contain sequences that do not serve a specificfunction in the target cell but are used in the generation of thepolynucleotide. Such sequences include, but are not limited to,sequences required for replication or selection of the polynucleotide ina host organism.

The term gene generally refers to a polynucleotide sequence thatcomprises coding sequences necessary for the production of a therapeuticpolynucleotide (e.g., ribozyme) or a polypeptide or precursor. Thepolypeptide can be encoded by a full length coding sequence or by anyportion of the coding sequence so long as the desired activity orfunctional properties (e.g., enzymatic activity, ligand binding, signaltransduction) of the full-length polypeptide or fragment are retained.The term also encompasses the coding region of a gene and the includingsequences located adjacent to the coding region on both the 5′ and 3′ends for a distance of about 1 kb or more on either end such that thegene corresponds to the length of the full-length mRNA. The sequencesthat are located 5′ of the coding region and which are present on themRNA are referred to as 5′ untranslated sequences. The sequences thatare located 3′ or downstream of the coding region and which are presenton the mRNA are referred to as 3′ untranslated sequences. The term geneencompasses both cDNA and genomic forms of a gene. A genomic form orclone of a gene contains the coding region interrupted with non-codingsequences termed introns, intervening regions or intervening sequences.Introns are segments of a gene which are transcribed into nuclear RNA.Introns may contain regulatory elements such as enhancers. Introns areremoved or spliced out from the nuclear or primary transcript; intronstherefore are absent in the mature RNA transcript. The messenger RNA(mRNA) functions during translation to specify the sequence or order ofamino acids in a nascent polypeptide. A gene may also includes otherregions or sequences including, but not limited to, promoters,enhancers, transcription factor binding sites, polyadenylation signals,internal ribosome entry sites, silencers, insulating sequences, matrixattachment regions. These sequences may be present close to the codingregion of the gene (within 10,000 nucleotides) or at distant sites (morethan 10,000 nucleotides). These non-coding sequences influence the levelor rate of transcription and/or translation of the gene. Covalentmodification of a gene may influence the rate of transcription (e.g.,methylation of genomic DNA), the stability of mRNA (e.g., length of the3′ polyadenosine tail), rate of translation (e.g., 5′ cap), nucleic acidrepair, nuclear transport, and immunogenicity. One example of covalentmodification of nucleic acid involves the action of LABELIT® reagents(Mirus Corporation, Madison, Wis.).

As used herein, the term gene expression refers to the process ofconverting genetic information encoded in a gene into RNA (e.g., smallRNA, siRNA, mRNA, rRNA, tRNA, or snRNA) through transcription of adeoxyribonucleic gene (e.g., via the enzymatic action of an RNApolymerase), and for protein encoding genes, into protein throughtranslation of mRNA. Gene expression can be regulated at many stages inthe process. Up-regulation or activation refers to regulation thatincreases the production of gene expression products (i.e., RNA orprotein), while down-regulation or repression refers to regulation thatdecrease production. Molecules (e.g., transcription factors) that areinvolved in up-regulation or down-regulation are often called activatorsand repressors, respectively.

The polynucleotide may be used for research purposes or to produce achange in a cell that can be therapeutic. The delivery of apolynucleotide for therapeutic purposes is commonly called gene therapy.The delivery of a polynucleotide can lead to modification of the geneticmaterial present in the target cell. The term stable transfection orstably transfected generally refers to the introduction and integrationof an exogenous polynucleotide into the genome of the transfected cell.The term stable transfectant refers to a cell which has stablyintegrated the polynucleotide into the genomic DNA. Stable transfectioncan also be obtained by using episomal vectors that are replicatedduring the eukaryotic cell division (e.g., plasmid DNA vectorscontaining a papilloma virus origin of replication, artificialchromosomes). The term transient transfection or transiently transfectedrefers to the introduction of a polynucleotide into a cell where thepolynucleotide does not integrate into the genome of the transfectedcell. If the polynucleotide contains an expressible gene, then theexpression cassette is subject to the regulatory controls that governthe expression of endogenous genes in the chromosomes. The termtransient transfectant refers to a cell which has taken up apolynucleotide but has not integrated the polynucleotide into itsgenomic DNA.

Formulation

The polynucleotide-polymer conjugate is formed by covalently linking thepolynucleotide to the polymer. The polymer is polymerized or modifiedsuch that it contains a reactive group A. The polynucleotide is alsopolymerized or modified such that it contains a reactive group B.Reactive groups A and B are chosen such that they can be linked via areversible covalent linkage using methods known in the art.

Conjugation of the polynucleotide to the polymer can be performed in thepresence of an excess of polymer. Because the polynucleotide and thepolymer may be of opposite charge during conjugation, the presence ofexcess polymer can reduce or eliminate aggregation of the conjugate.Alternatively, an excess of a carrier polymer, such as a polycation, canbe used. The excess polymer can be removed from the conjugated polymerprior to administration of the conjugate to the animal or cell culture.Alternatively, the excess polymer can be co-administered with theconjugate to the animal or cell culture.

Similarly, the polymer can be conjugated to the masking agent in thepresence of an excess of polymer or masking agent. Because thepolynucleotide and the polymer may be of opposite charge duringconjugation, the presence of excess polymer can reduce or eliminateaggregation of the conjugate. Alternatively, an excess of a carrierpolymer can be used. The excess polymer can be removed from theconjugated polymer prior to administration of the conjugate to theanimal or cell culture. Alternatively, the excess polymer can beco-administered with the conjugate to the animal or cell culture. Thepolymer can be modified prior to or subsequent to conjugation of thepolynucleotide to the polymer.

Transfection Reagent

The process of delivering a polynucleotide to a cell has been commonlytermed transfection or the process of transfecting. The termtransfecting as used herein refers to the introduction of apolynucleotide or other biologically active compound from outside a cellto inside cell such the polynucleotide has biologically activity. Thepolynucleotide may be used for research purposes or to produce a changein a cell that can be therapeutic. The delivery of a polynucleotide canlead to modification of the genetic material present in the target cell.A transfection reagent or delivery vehicle is a compound or compoundsthat bind(s) to or complex(es) with oligonucleotides andpolynucleotides, and mediates their entry into cells.

In vitro transfection reagents, or delivery vehicles, are compounds orcompositions of compounds that bind to or complex with oligonucleotidesand polynucleotides and mediate their entry into cells. Examples oftransfection reagents include, but are not limited to, protein andpolymer complexes (polyplexes), lipids and liposomes (lipoplexes),combinations of polymers and lipids (lipopolyplexes), calcium phosphateprecipitates, and dendrimers. Typically, the transfection reagent has acomponent with a net positive charge that binds to the oligonucleotide'sor polynucleotide's negative charge. Cationic transfection agents mayalso condense large nucleic acids. Transfection agents may also be usedto associate functional groups with a polynucleotide. Functional groupsinclude cell targeting signals, nuclear localization signals, compoundsthat enhance release of contents from endosomes or other intracellularvesicles (such as membrane active compounds), and other compounds thatalter the behavior or interactions of the compound or complex to whichthey are attached (interaction modifiers).

The conjugated polynucleotides of the instant invention provide usefulreagents and methods for a variety of therapeutic, diagnostic, targetvalidation, genomic discovery, genetic engineering and pharmacogenomicapplications. The conjugated polynucleotide has improved cellular uptakeproperties compared with the same unconjugated polynucleotide.

Parenteral routes of administration include intravascular (intravenous,intraarterial), intramuscular, intraparenchymal, intradermal, subdermal,subcutaneous, intratumor, intraperitoneal, intrathecal, subdural,epidural, and intralymphatic injections that use a syringe and a needleor catheter. Intravascular herein means within a tubular structurecalled a vessel that is connected to a tissue or organ within the body.Within the cavity of the tubular structure, a bodily fluid flows to orfrom the body part. Examples of bodily fluid include blood,cerebrospinal fluid (CSF), lymphatic fluid, or bile. Examples of vesselsinclude arteries, arterioles, capillaries, venules, sinusoids, veins,lymphatics, bile ducts, and ducts of the salivary or other exocrineglands. The intravascular route includes delivery through the bloodvessels such as an artery or a vein. The blood circulatory systemprovides systemic spread of the pharmaceutical. An administration routeinvolving the mucosal membranes is meant to include nasal, bronchial,inhalation into the lungs, or via the eyes. Intraparenchymal includesdirect injection into a tissue such as liver, lung, heart, muscle(skeletal muscle or diaphragm), spleen, pancreas, brain (includingintraventricular), spinal cord, ganglion, lymph nodes, adipose tissues,thyroid tissue, adrenal glands, kidneys, prostate, and tumors.Transdermal routes of administration have been affected by patches andiontophoresis. Other epithelial routes include oral, nasal, respiratory,rectum, and vaginal routes of administration.

The conjugates can be injected in a pharmaceutically acceptable carriersolution. Pharmaceutically acceptable refers to those properties and/orsubstances which are acceptable to the mammal from apharmacological/toxicological point of view. The phrase pharmaceuticallyacceptable refers to molecular entities, compositions, and propertiesthat are physiologically tolerable and do not typically produce anallergic or other untoward or toxic reaction when administered to amammal. Preferably, as used herein, the term pharmaceutically acceptablemeans approved by a regulatory agency of the Federal or a stategovernment or listed in the U.S. Pharmacopeia or other generallyrecognized pharmacopeia for use in animals, and more particularly inhumans.

Therapeutic Effect

We have disclosed polynucleotide delivery, resulting in gene expressionor inhibition of gene expression of reporter genes and endogenous genesin specific tissues. Levels of a reporter (marker) gene expressionmeasured following delivery of a polynucleotide indicate a reasonableexpectation of similar levels of gene expression following delivery ofother polynucleotides. Levels of treatment considered beneficial by aperson having ordinary skill in the art differ from disease to disease,for example: Hemophilia A and B are caused by deficiencies of theX-linked clotting factors VIII and IX, respectively. Their clinicalcourse is greatly influenced by the percentage of normal serum levels offactor VIII or IX: <2%, severe; 2-5%, moderate; and 5-30% mild. Thus, anincrease from 1% to 2% of the normal level of circulating factor insevere patients can be considered beneficial. Levels greater than 6%prevent spontaneous bleeds but not those secondary to surgery or injury.Similarly inhibition of a gene need not be 100% to provide a therapeuticbenefit. A person having ordinary skill in the art of gene therapy wouldreasonably anticipate beneficial levels of expression of a gene specificfor a disease based upon sufficient levels of marker gene results. Inthe hemophilia example, if marker genes were expressed to yield aprotein at a level comparable in volume to 2% of the normal level offactor VIII, it can be reasonably expected that the gene coding forfactor VIII would also be expressed at similar levels. Thus, reporter ormarker genes such as the genes for luciferase and β-galactosidase serveas useful paradigms for expression of intracellular proteins in general.Similarly, reporter or marker genes secreted alkaline phosphatase (SEAP)serve as useful paradigms for secreted proteins in general.

A therapeutic effect of the protein expressed from a deliveredpolynucleotide in attenuating or preventing a disease, condition, orsymptom can be accomplished by the protein either staying within thecell, remaining attached to the cell in the membrane or being secretedand dissociating from the cell where it can enter the generalcirculation and blood. Secreted proteins that can be therapeutic includehormones, cytokines, growth factors, clotting factors, anti-proteaseproteins (e.g. alpha-antitrypsin) and other proteins that are present inthe blood. Proteins on the membrane can have a therapeutic effect byproviding a receptor for the cell to take up a protein or lipoprotein.For example, the low density lipoprotein (LDL) receptor could beexpressed in hepatocytes and lower blood cholesterol levels and therebyprevent atherosclerotic lesions that can cause strokes or myocardialinfarction. Therapeutic proteins that stay within the cell can beenzymes that clear a circulating toxic metabolite as in phenylketonuria.They can also cause a cancer cell to be less proliferative or cancerous(e.g. less metastatic). A protein within a cell could also interferewith the replication of a virus.

Liver is one of the most important target tissues for gene therapy givenits central role in metabolism (e.g., lipoprotein metabolism in varioushypercholesterolemias) and the secretion of circulating proteins (e.g.,clotting factors in hemophilia). At least one hundred different geneticdisorders could potentially be corrected by liver-directed gene therapy.In addition, acquired disorders such as chronic hepatitis and cirrhosisare common and could also be treated by polynucleotide-based livertherapies. Gene therapies involving heterotropic gene expression wouldfurther enlarge the number of disorders treatable by liver-directed genetransfer. For example, diabetes mellitus could be treated by expressingthe insulin gene within hepatocytes whose physiology may enableglucose-regulated insulin secretion.

In addition to increasing or decreasing genes involved in metabolism ordisease, delivery of polynucleotides to cell in vivo can also be used tostimulate the immune system, stimulate the immune system to destroy thecancer cells, inactive oncogenes, or activate or deliver tumorsuppressor genes. Polynucleotides can also be delivered to induceimmunity to pathogens or to block expression of pathogenic genes.

Miscellaneous Definitions

As used herein, in vivo means that which takes place inside an organismand more specifically to a process performed in or on the living tissueof a whole, living multicellular organism (animal), such as a mammal, asopposed to a partial or dead one.

As used herein, in vitro refers to a process performed in an artificialenvironment created outside a living multicellular organism (e.g., atest tube or culture plate) used in experimental research to study adisease or process. As used herein, in vitro includes processesperformed in intact cells growing in culture.

As used herein, in situ refers to processes carried out in intacttissue. However, the tissue can be in a living organism or removed fromthe organism.

As used herein, ex vivo refers to a process performed in an artificialenvironment outside the organism on living cells or tissue which areremoved from an organism and subsequently returned to an organism.

Crosslinkers are bifunctional molecules used to connect two moleculestogether, i.e. form a linkage between two molecules. Bifunctionalmolecules can contain homo or heterobifunctionality.

As used herein, a surface active polymer lowers the surface tension ofwater and/or the interfacial tension with other phases, and,accordingly, is positively adsorbed at the liquid/vapor interface.

An antibody is any immunoglobulin, including antibodies and fragmentsthereof, that binds a specific epitope. The term encompasses polyclonal,monoclonal, and chimeric antibodies. An antibody combining site is thatstructural portion of an antibody molecule comprised of heavy and lightchain variable and hypervariable regions that specifically bindsantigen. The phrase antibody molecule in its various grammatical formsas used herein contemplates both an intact immunoglobulin molecule andan immunologically active portion of an immunoglobulin molecule.Exemplary antibody molecules are intact immunoglobulin molecules,substantially intact immunoglobulin molecules and those portions of animmunoglobulin molecule that contains the paratope, including thoseportions known in the art as Fab, Fab′, F(ab′).sub.2 and F(v), whichportions are preferred for use in the therapeutic methods describedherein. Fab and F(ab′).sub.2 portions of antibody molecules are preparedby the proteolytic reaction of papain and pepsin, respectively, onsubstantially intact antibody molecules by methods that are well-known.The phrase monoclonal antibody in its various grammatical forms refersto an antibody having only one species of antibody combining sitecapable of immunoreacting with a particular antigen. A monoclonalantibody thus typically displays a single binding affinity for anyantigen with which it immunoreacts. A monoclonal antibody may thereforecontain an antibody molecule having a plurality of antibody combiningsites, each immunospecific for a different antigen; e.g., a bispecific(chimeric) monoclonal antibody.

EXAMPLES Example 1 Polynucleotide Synthesis and Assembly

The following synthetic RNA oligonucleotides (Dharmacon, LafayetteColo.) were used. Sense strand RNAs contained a primary amine with a sixcarbon spacer at the 5′ to allow conjugation to the delivery vehicle.The 2′ACE protected RNA oligonucleotides were deprotected prior to theannealing step according to the manufacturer's instructions. The siRNAshad the following sequences:

apoB (Ensembl# ENSMUST00000037520); apoB-1 siRNA sense5′-NH₄-GAAmUGmUGGGmUGGmCAAmCmUmUmUmA*G,, SEQ ID 1 antisense5′-P-AmAAGUUGCCACCCACAUUCmA*G,; SEQ ID 2 apoB-2 siRNA sense5′-NH₄-GGAmCAmUGGGmUmUCCAAAmUmUAmC*G,, SEQ ID 3 antisense5′-P-UmAAUUUGGAACCCAUGUCCmC*G,; SEQ ID 4 ppara (GenBank# NM_011144);ppara-1 siRNA, sense 5′-NH₄-mUmCAmCGGAGmCmUmCAmCAGAAmUmUmC*U-3′,, SEQ ID5 antisense 5′-P-AmAUUCUGUGAGCUCCGUGAmC*U-3′,; SEQ ID 6 ppara-2 siRNAsense 5′-NH₄-mUmCCCAAAGCmUCCmUmUmCAAAAmU*U-3′,, SEQ ID 7 antisense5′-P-mUmUUUGAAGGAGCUUUGGGAmA*G-3′,; SEQ ID 8 Control siRNA (GL-3luciferase reporter gene), sense5′-NH₄-mCmUmUAmCGmCmUGAGmUAmCmUmUmCGAmU*U-3′;, SEQ ID 9 antisense5′-P-UmCGAAGUACUCAGCGUAAGmU*U;; SEQ ID 10 m = 2′-O-CH₃ substitution, *= phosphorothioate linkage, and P = PO₄.

Sense and antisense oligonucleotides for each target sequence wereannealed by mixing equimolar amounts of each and heating to 94° C. for 5min, cooling to 90° C. for 3 min, then decreasing the temperature in0.3° C. steps 250 times, holding at each step for 3 sec.

Example 2 Polyvinylether Random Copolymers

A. Synthesis of a vinyl ether monomer. 2-Vinyloxy Ethyl Phthalimide wasprepared via reacting 2-chloroethyl vinyl ether (25 g, 0.24 mol) andpotassium phthalimide (25 g, 0.135 mol) in 100° C. DMF (75 ml) usingtetra n-butyl ammonium bromide (0.5 g) as the phase transfer catalyst.This solution was heated for 6 h and then crashed out in water andfiltered. This solid was then recrystallized twice from methanol to givewhite crystals.

B. Amine+lower alkyl polyvinylether polymers. A series of copolymers wassynthesized from vinylether monomers with varying alkyl to amine groupratios and with alkyl groups having from one to four carbons (FIG. 1, Rand R′ may be the same or different). Membrane activity was dependent onthe size (alkyl chain length) and ratio of hydrophobic monomers(Wakefield 2005). Propyl and butyl-derived polymers were found to bemembrane lytic using model liposomes while methyl and ethyl containingpolymers were not. We termed these polymers methyl, ethyl, propyl, orbutyl-aminovinyl ethers or (PMAVE, PEAVE, PPAVE, or PBAVE respectively).These polymers possess sufficient charge to complex with DNA inphysiological concentrations of salt. In contrast, small membrane lyticcationic peptides such as melittin are too weak and cannot condense DNAin isotonic saline solutions. The larger size of the syntheticamphipathic polymers not only enhances their DNA binding ability, butalso makes them more lytic on a molar basis than melittin. The abilityto lyse membranes with less number of polymer molecules will facilitatein vivo nucleic acid delivery. Using fluorophore-labeled DNA, wedetermined the charge density of the synthesized polymers and confirmedtheir stability in saline.

C. Synthesis of water-soluble, amphipathic, membrane activepolyvinylether polyamines terpolymers. X mol % amine-protectedvinylether (e.g., 2-Vinyloxy Ethyl Phthalimide) was added to an ovendried round bottom flask under a blanket of nitrogen in anhydrousdichloromethane. To this solution Y mol % alkyl (e.g., ethyl, propyl, orbutyl) vinylether was added, followed by Z mol % alkyl(dodecyl,octadecyl) vinylether (FIG. 1). While the polymers detailed are derivedfrom 2-3 different monomers, the invention is not limited to a specificcomposition of vinyl ether monomers. Polymers comprising more monomersor different monomers were readily envisioned. The solution was broughtto −78° C. in a dry ice acetone bath. To this solution 10 mol % BF₃EtOEtwas added and the reaction was allowed to proceed for 2-3 h at −78° C.,and then quenched with a methanol ammonium hydroxide solution. Thepolymer was brought to dryness under reduced pressure and then broughtup in 30 ml of 1,4-dioxane/methanol (2/1). 20 mol eq. of hydrazine perphthalimide was added to remove the protecting group from the amine. Thesolution was refluxed for 3 h, then brought to dryness under reducedpressure. The solid was brought up in 20 ml 0.5 M HCl, refluxed for 15min, diluted with 20 ml distilled water, and refluxed for additionalhour. The solution was then neutralized with NaOH, cooled to roomtemperature, transferred to 3,500 molecular cellulose tubing, dialyzedfor 24 hrs (2×20 L) against distilled water, and freeze dried. Themolecular weight of the polymers was estimated using analytical sizeexclusion columns according to standard procedures. While polymerscontaining the indicated vinyl ether monomers are described in theseexamples, the invention is not limited to these particular monomers.

After removal of the phthalimide groups by sequential treatment withhydrazine and HCl, the polymers were transfer to 3,500 molecularcellulose tubing and dialyzed for 24 h (2×20 L) against distilled water,and freeze dried. The polymers were then dissolved in water and placedonto a sephadex G-15 column. The polymers that were excluded fromsephadex G-15 were isolated and concentrated by lyophilization. Themolecular weights of the polymers were then determined by GPC usingEprogen Inc. CATSEC100, CATSEC300, and CATSEC1000 columns in series. Therunning buffer was 50 mM NaCl, 0.1% TFA, and 10% MeOH. Polyvinylpyridinestandards were used as the calibration curve. The molecular weights ofthe polymers were in the range 10,000-100,000 Da, with the majority ofpolymer preparations over 20,000 Da.

D. Synthesis of DW1360. An amine/butyl/octadecyl polyvinyletherterpolymer, was synthesized from 2-Vinyloxy Ethyl Phthalimide (3.27 g,15 mmol), butyl vinylether (0.40 g, 4 mmol), and octadecylvinylether(0.29 g, 1 mmol) monomers. The monomers were dissolved in 30 mlanhydrous dichloromethane. These solutions were then added to a −78° C.dry ice/acetone bath. 2 min later, BF₃.OEt₂ (0.042 g, 0.3 mmol) wasadded and the reaction was allowed to proceed for 3 h at −78° C. Thepolymerization was then stopped by the addition of 50/50 mixture ofammonium hydroxide in methanol or a solution of lithium borohydride. Thesolvents were then removed by rotary evaporation. The polymer was thendissolved in 30 ml of 1,4-dioxane/methanol (2/1). To this solution wasadded hydrazine (0.44 g, 138 mmol) and the mixture was heated to refluxfor 3 h. The solvents were then removed by rotary evaporation and theresulting solid was then brought up in 20 ml of 0.5 M HCl and refluxedfor 15 min, diluted with 20 ml distilled water, and refluxed foradditional hour. This solution was then neutralized with NaOH, cooled toRT, transferred to 3,500 molecular cellulose tubing, and dialyzed for 24h (2×20 L) against distilled water, and lyophilized.

Similarly, other polymers containing various ratios of amine, butyl andoctadecyl groups may be synthesized. Also other monomers may beincorporated. In particular, t-butyl vinyl ether, dodecyl vinylether,and oleic vinylether have been incorporated into polymers.

In one embodiment, the amine/lower alkyl/higher alkyl polyvinyletherterpolymers can be synthesized using monomers at a feed ratio of 15amine monomer:4 lower alkyl monomer:1 higher alkyl monomer. Under theconditions described above, the incorporation ratio was determined to beabout 5.4-7.5 amine monomer:3-3.5 lower alkyl monomer:1 higher alkylmonomer. In another embodiment, the amine/lower alkyl/higher alkylpolyvinylether terpolymers are synthesized using monomers at a feedratio of 4-8 amine monomer:3-5 lower alkyl monomer:1 higher alkylmonomer. In a preferred embodiment, the polymers are water soluble(about 1 mg/ml or greater at 25° C.) and surface active.

In one embodiment, the polymers are fractionated to yield polymers ofmolecule weight of about 5 kDa to about 50 kDa, and more preferablyabout 10 kDa to about 30 kDa.

E. Other terpolymers. Similar polymers can be synthesized ofmodification of different base polymers, including, but not limited to:poly-L-lysine (PLL), polyvinylamine (PVA), and polyallylamine (PAA). Byattachment of alkyl groups to different the main chain of thesepolymers, terpolymers can by made with a ratio of amine:loweralkyl:higher alkyl of about 6:3:1.

F. Liposome lysis. 10 mg of egg phosphatidylcholine was hydrated with 1ml of buffer containing 100 mM carboxyfluorescein (CF) and 10 mM HEPESpH 7.5. Liposomes were then be extruded through 100-nm porespolycarbonate filters (Nucleopore, Pleasanton, Calif.). Unentrapped CFwas removed by size exclusion chromatography using Sepharose 4B-200eluting with 10 mM HEPES pH 8.0.1 M NaCl. A 200 μL aliquot of theCF-loaded liposomes were added to 1.8 ml of isotonic buffer.Fluorescence (λ_(ex)=488, λ_(em)=540) was measured 30 min after additionof 0.25 μg of polymers or melittin to vesicle suspensions. At the end ofeach experiment, vesicles were disrupted by the addition of 40 μl of a1% Triton X-100 solution to determine maximal lysis.

Example 3 Masking Agents

A. Synthesis of 2-propionic-3-methylmaleic anhydride(carboxydimethylmaleic anhydride or CDM). To a suspension of sodiumhydride (0.58 g, 25 mmol) in 50 ml anhydrous tetrahydrofuran was addedtriethyl-2-phosphonopropionate (7.1 g, 30 mmol). After evolution ofhydrogen gas had stopped, dimethyl-2-oxoglutarate (3.5 g, 20 mmol) in 10ml anhydrous tetrahydrofuran was added and stirred for 30 min. Water, 10ml, was then added and the tetrahydrofuran was removed by rotaryevaporation. The resulting solid and water mixture was extracted with3×50 ml ethyl ether. The ether extractions were combined, dried withmagnesium sulfate, and concentrated to a light yellow oil. The oil waspurified by silica gel chromatography elution with 2:1 ether:hexane toyield 4 g (82% yield) of pure triester. The 2-propionic-3-methylmaleicanhydride was then formed by dissolving of this triester into 50 ml of a50/50 mixture of water and ethanol containing 4.5 g (5 equivalents) ofpotassium hydroxide. This solution was heated to reflux for 1 h. Theethanol was then removed by rotary evaporation and the solution wasacidified to pH 2 with hydrochloric acid. This aqueous solution was thenextracted with 200 ml ethyl acetate, which was isolated, dried withmagnesium sulfate, and concentrated to a white solid. This solid wasthen recrystallized from dichloromethane and hexane to yield 2 g (80%yield) of 2-propionic-3-methylmaleic anhydride.

Thioesters, esters and amides may be synthesized from CDM by conversionof CDM to its acid chloride with oxalyl chloride followed by theaddition of a thiol, ester or amine and pyridine. CDM and itsderivatives are readily modified by methods standard in the art, to addtargeting ligands, steric stabilizers, charged groups, and otherreactive groups. The resultant molecules can be used to reversiblymodify amines.

B. Galactose-containing targeting groups. The most widely-studiedhepatocyte targeting ligands are based on galactose, which is bound bythe asialoglycoprotein receptor (ASGPr) on hepatocytes. Attachment ofgalactose has been shown to facilitate hepatocyte targeting of a fewhighly water soluble, uncharged polymers, including: the oligosaccharidechitosan, a polystyrene derivative, and a polyacrylamide HPMA. Galactosetargeting groups are readily generated using lactose, agalactose-glucose disaccharide, via modification of the glucose residue.Lactobionic acid (LBA, a lactose derivative in which the glucose hasbeen oxidized to gluconic acid) is readily incorporated into a maleicanhydride derivative using standard amide coupling techniques. FIG. 2shows the structure of two LBA derivatives that couple galactose to themaleic anhydride CDM.

Maleamate modification converts a positively-charged amine group into anegatively charged one. This charge modification can reduce non-specificinteraction of the polymer with negatively charged cells and serumcomponents. However, too much negative charge can enhance interactionwith scavenger receptors. A net neutral galactose-containing maleicanhydride derivative can be generated by inserted a positively chargedtertiary amine group into the masking agent, creating a zwitterion. Sucha compound, CDM-Pip-LBA, was generated from the components: CDM,propylaminopiperazine, and lactobionic acid (FIG. 2).

C. Steric stabilizer CDM-PEG and targeting group CDM-NAG (N-acetylgalactosamine) syntheses (FIG. 2). To a solution of CDM (Rozema 2003)(300 mg, 0.16 mmol) in 50 ml methylene chloride was added oxalylchloride (2 g, 10 wt. eq.) and dimethylformamide (5 μl). The reactionwas allowed to proceed overnight at which time the excess oxalylchloride and methylene chloride were removed by rotary evaporation toyield the CDM acid chloride. The acid chloride was dissolved in 1 ml ofmethylene chloride. To this solution was added 1.1 molar equivalentspolyethylene glycol monomethyl ether (MW average 450) for CDM-PEG or(aminoethoxy)ethoxy-2-(acetylamino)-2-deoxy-β-D-glucopyranoside (i.e.amino bisethoxylethyl NAG) for CDM-NAG, and pyridine (200 μl, 1.5 eq) in10 ml of methylene chloride. The solution was then stirred 1.5 h. Thesolvent was then removed and the resulting solid was dissolved into 5 mlof water and purified using reverse-phase HPLC using a 0.1% TFAwater/acetonitrile gradient (FIG. 2).

Example 4 Reversible Attachment of Polynucleotide to Polymer

Modification of amino siRNA oligos with S-acetyl groups resulted instable protected thiols that could be released in a basic solution(pH≧9) in the presence of an amine-containing polycation. Standarddeprotection conditions for removal of a thioester group requireincubation with hydroxylamine. However, these deprotection conditionsresult in significant siRNA disulfide dimer formation. Typically, thereaction between an alkyl amine and thioester is relatively slow, but ina complex between polyanionic siRNA and a polycationic amine, thereaction between functional groups became essentially intramolecular andwas greatly accelerated (see Step 2a in FIG. 3). In addition toaccelerating the deprotection, the released thiol's reaction withactivated disulfide groups on the polycation was greatly enhanced (Step2b). The activated disulfide pyridyldithiol (PD) insured selectivedisulfide formation, and was easily attached to the polycation usingcommercially available reagents. The result of these intra-particlereactions was >90% conjugation of disulfide to polycation.

Example 5 Reversible Conjugation of Polynucleotide to Polymer

A. SATA modified polynucleotide. N-succinimidyl-5-acetylthioacetate(SATA)-modified polynucleotides were synthesized by reaction of 5′amine-modified siRNA with 1 weight equivalents (wt. eq.) of SATA reagent(Pierce) and 0.36 wt. eq. of NaHCO₃ in water at 4° C. for 16 h. Theprotected thiol modified siRNAs were precipitated by the addition of 9volumes of ethanol and incubation at −78° C. for 2 h. The precipitatewas isolated, dissolved in 1×siRNA buffer (Dharmacon), and quantitatedby measuring absorbance at the 260 nm wavelength.

Polymer, in this example PBAVE or DW1360 (30 mg/ml in 5 mM TAPS pH 9),was modified by addition of 1.5 wt %4-succinimidyloxycarbonyl-α-methyl-α-[2-pyridyldithio]-toluene (SMPT,Pierce). 1 h after addition of SMPT, 0.8 mg of SMPT-polymer was added to400 μl isotonic glucose solution containing 5 mM TAPS pH 9. To thissolution was added 50 μg of SATA-modified siRNA. For the dose responseexperiments where PBAVE concentration was constant, different amounts ofsiRNA were added. The mixture was then incubated for 16 h. In this way,the polynucleotide was conjugated to the polymer via a reversibledisulfide bone. A disulfide bond for conjugation between the polymer andsiRNA provides for reversibility in the reducing environment of thecytoplasm. The siRNA-polymer conjugate was masked by adding to thesolution 5.6 mg of HEPES free base followed by a mixture of 3.7 mgCDM-NAG and 1.9 mg CDM-PEG. The solution was then incubated 1 h at roomtemperature (RT) before injection.

B. Quantitation of conjugated polynucleotide. To quantify the amount ofconjugated siRNA, 10 μl aliquots of siRNA-polymer conjugate containing 1μg siRNA were treated with 2 μl of 1 M DTT or with no additive at RT for16 h. 100 μg polyacrylic acid and 300 μg NaCl were added to the samplesto neutralize electrostatic interactions. After a 2 h incubation, thesamples were electrophoresed on a 2% agarose gel and the siRNAvisualized by staining with ethidium bromide. The siRNA bands werequantified using Kodak Molecular Imaging Software v4.0. The amount ofsiRNA unconjugated was normalized to the amount released upon DTTtreatment to determine percent conjugated. The amount of conjugatedsiRNA was typically 70-90%.

Other disulfide conjugation chemistry may be used wherein the thiol isprotected as a disulfide or not protected at all. Additionally, thepolymer may be modified with other activated disulfide groups, thiolgroups, or protected thiol groups.

Example 6 Reversible Attachment of CDM-Masking Agents toPolynucleotide-Polymers Conjugates

The polynucleotide-polymer conjugates may be modified, such as withmaleic acid derivatives, to reversibly modify the polymer (FIG. 3, Step4.). The siRNA-polymer conjugate was masked by adding to the solution5.6 mg of HEPES free base followed by a mixture of 3.7 mg CDM-NAG and1.9 mg CDM-PEG. The solution was then incubated 1 h at room temperature(RT) before in vivo injection.

Example 7 Targeting Activity of Masked Conjugates

To demonstrate the ability of CDM-PIP-LBA (galactose-containing) agentsto enable liver targeting of the amphipathic polyvinylether 25/75 (25:75butyl:amine) PBAVE, the polymer was labeled with the fluorophore Cy3followed by modification with CDM-PEG and with or without CDM-Pip-LBA.100 μg masked polymer in 400 μl isotonic glucose was injected into thetail vein of mice. Liver samples were removed 1 h after injection andfrozen liver sections were prepared and processed forimmunohistochemistry (FIG. 4). As shown in FIG. 4, CDM-Pip-LBA targetedpolymer was extensively located within hepatocytes (B) whereas theuntargeted polymer was localized primarily with macrophages of the liver(A).

CDM-LBA modified PBAVE is highly anionic and was found primarily withmacrophages (not shown). CDM-Pip-LBA (zwitterionic masking agent)modified polymers were near neutral and exhibited an increase in theamount of polymer targeted to the liver as a whole and, moreimportantly, to hepatocytes in particular (FIG. 4B). It is also possibleto shield charge, such as negative charge, with steric stabilizers,including CDM-PEG masking agents.

Example 8 Targeting of Polynucleotide to Hepatocytes

An oligonucleotide-polymer conjugate was made as described above andmasked with CDM-Pip-LBA. 20 μg of SATA/Cy3 modified siRNA was conjugatedto 1.8 mg of PD-modified PLL and modified with CDM-Pip-LBA. The maskedconjugate was injected in 400 μL of saline into the tail vein of amouse. In the same injection solution was 100 μg of CDM-Pip-LBA-modified25/75-PBAVE labeled with Oregon Green. The conjugates were soluble andnonaggregating in physiological conditions. 1 h postinjection, the liverwas harvested and frozen liver sections were prepared and processed forimmunohistochemistry. TO-PRO-3 was used to stain cell nuclei. Afterinjection of into the tail vein of mice, labeled oligonucleotide andPBAVE were observed in hepatocytes (top left panel of FIG. 5).

Example 9 In Vivo Activity of Conjugate

The ability of the masked polynucleotide-polymer conjugates to deliversiRNA for the inhibition of an endogenous gene in mouse liver,peroxisome proliferator activated receptor alpha (PPARa), was assessed.C57B mice (n=4) were injected with CDM-Pip-LBA modified PPARa siRNA-PLLconjugate (20 μg siRNA, 1.8 mg PD-PLL) and 25/75-PBAVE (100 μg) in 400μL of isotonic glucose via the tail vein. RNA was prepared from liver 48h after injection. PPARa mRNA levels were determined using aquantitative real-time PCR (qRT-PCR). mRNA levels were normalized toanimals injected with anti-luciferase siRNA. Changes to mRNA levels werenormalized to endogenous GAPDH. Three independent experiments wereperformed. As shown in FIG. 6, a 20% inhibition of PPARa mRNA levels wasobtained.

Example 10 In Vivo Delivery/Activity of Conjugate

Six to eight week old male mice (strain C57BL/6, 20-25 g) were obtainedfrom Harlan Sprague Dawley (Indianapolis, Ind.). Mice were housed atleast 10 days prior to injection. Feeding was performed ad libitu withHarlan Teklad Rodent Diet (Harlan, Madison Wis.). Mice were infused withinto the tail vein in a total volume of 0.4 ml injection HEPES-buffered(5 mM pH 7.5) isotonic glucose. Mice were injected with either DNA orsiRNA conjugates.

Mice were fasted for 4 h prior to serum collection by retro-orbitalbleed and liver harvest. Serum for use in Western assays was collectedand added to an equal volume of Complete Protease Inhibitor Cocktailcontaining EDTA (Roche, Indianapolis Ind.) and stored at −20° C. TotalRNA was isolated from liver immediately after harvest using TRI-REAGENT®according to the manufacturer's protocol (Molecular Research Center,Cincinnati Ohio).

For analysis of hepatocyte targeting, polyconjugate containing 10 μg ofCy3-labeled, 21-mer dsDNA was formulated as described above in a totalvolume of 0.2 ml and delivered into male ICR mice (20-25 g, HarlanSprague Dawley) by i.v. injection. 1 h after injection, mice weresacrificed and liver samples were excised. In some experiments, tissuesamples from lung, kidney, spleen, brain and pancreas were also excised.Tissue samples were fixed in 4% paraformaldehyde/PBS for 6 h and thenplaced into a 30% sucrose/PBS solution overnight at 4° C. Fixed tissuesamples were then placed into block holders containing OCT freezingmedium (Fisher Scientific, Pittsburgh, Pa.) and snap frozen in liquidnitrogen. Frozen tissue sections (8-10 μm) were prepared using a MicromHM 505N cryostat (Carl Zeiss, Thornwood, N.Y.) and placed ontoSuperfrost-Plus microscope slides (Fisher Scientific). Tissue sectionswere counterstained with ALEXA®-488 phalloidin (13 nM, Invitrogen,Carlsbad Calif.) and TO-PRO-3 (40 nM, Invitrogen) in PBS for 20 min. Theslides were mounted in Vectashield (Vector Laboratories, BurlingameCalif.) and analyzed by a LSM510 confocal microscope (Carl Zeiss).

Confocal micrographs of liver sections taken from mice 1 h afterinjection with polynucleotide-polymer conjugate containing aCy3-labeled, 21-mer double stranded DNA are shown in FIG. 7 (upper leftquadrant in each panel). Actin is shown in the upper right quadrants toindicate cell outlines. Nuclei are shown in the lower left quadrants.Merged pictures are shown in the lower right quadrants. Accumulation ofthe Cy3-labeled dsDNA in hepatocytes was observed when the polymercontained the NAG targeting moiety, with minimal uptake by Kupffer cellsor accumulation in liver sinusoids (FIG. 7A). Distribution was nearlyhomogenous throughout the different lobes of the liver. Inspection ofother organs revealed minor Cy3-fluorescence in spleen and kidney, atlevels estimated to be at least 20-fold lower than in liver. Injectionof unconjugated polynucleotide+polymer or replacement of CDM-NAG on thedelivery vehicle with CDM-glucose resulted in markedly reducedhepatocyte uptake and increased uptake by spleen and kidney (FIGS. 7Band 7C, respectively), which is consistent with glucose's low affinityfor the asialoglycoprotein receptor. Replacement of CDM-NAG on thedelivery vehicle with CDM-mannose resulted in increased macrophage andliver endothelial cell targeting (FIG. 7D).

Quantitative PCR and Invader Assays. In preparation for quantitativePCR, total RNA (500 ng) was reverse transcribed using SUPERSCRIPT III®(Invitrogen) and oligo-dT primers according to the manufacturer'sprotocol. Quantitative PCR was performed by using a Model 7500 FastReal-Time PCR system (Applied Biosystems, Foster City Calif.). TAQMAN®Gene Expression Assays for apoB and ppara were used in biplex reactionsin triplicate with GAPDH mRNA primers and probe using TAQMAN® UniversalPCR Master Mix (Applied Biosystems). The sequence of the GAPDH primersand probe (IDT, Coralville Iowa) were:GAPDH-forward 5′-AAATGGTGAAGGTCGGTGTG-3′,  SEQ ID 11;GAPDH-reverse 5′-CATGTAGTTGAGGTCAATGAAGG-3′,  SEQ ID 12;GAPDH-probe 5′-Hex/CGTGCCGCCTGGAGAAACCTGCC/BHQ-3′,  SEQ ID 13.

Direct measurements of ppara mRNA levels were performed using a customdesigned INVADER® mRNA assay according to the manufacturer'sinstructions (Third Wave Technologies, Madison Wis.). Biplex reactionswere performed in triplicate using the probe set for ubiquitin accordingto the manufacturer's instructions.

ApoB western, cytokine assays and liver toxicity and metabolic panels.Separation of serum proteins (0.1 μl) was accomplished byelectrophoresis on 3-8% polyacrylamide/SDS gels. The separated proteinswere electrophoretically transferred to PVDF membrane followed byincubation with a 1:5000 dilution of a rabbit polyclonal anti-ApoBantibody (Biodesign International, Saco Me.). The blot was thenincubated with a 1:80,000 dilution of goat anti-rabbit antibodyconjugated to horseradish peroxidase (Sigma), and antibody binding wasdetected using enhanced chemiluminescent detection kit (AmershamBiosciences, Piscataway N.J.). Serum levels of the mouse cytokines TNF-αand IL-6 were measured by sandwich ELISA using reagents according to themanufacturer's instructions (R&D Systems, Minneapolis Minn.). Serumlevels of mouse IFN-α were measured using a sandwich ELISA kit accordingto the manufacturer's instructions (PBL Biomedical, Piscataway N.J.).Serum levels of ALT, AST, cholesterol, and triglycerides were measuredusing automated systems at the Marshfield Clinic Laboratories VeterinaryDiagnostic Division (Marshfield Wis.).

Serum Levels of Cytokines and Liver Enzymes in siRNA Conjugate-TreatedMice. TNF-α IL-6 IFN-α ALT AST Treatment (pg/ml) (pg/ml) (pg/ml) (U/L)(U/L) Saline  6 h <6 <2 206 ± 51 60 ± 21 149 ± 21 48 h <6 <2 321 ± 77Control siRNA  6 h 7.9 ± 2.9 60.2 ± 2.4 207 ± 86 97 ± 26 176 ± 62 48 h<6  4.0 ± 1.3 211 ± 16 apoB-1 siRNA  6 h 57.9 ± 7.2  48.6 ± 2.5  494 ±165 71 ± 30 144 ± 34 48 h <6 <2 257 ± 70 n = 5, data are shown as mean ±s.d.

Example 11 Knockdown of Target Genes in Liver of Mice

To demonstrate the ability of the system to deliver siRNA and knockdowntarget gene expression in vivo, conjugate containing apoB-1 siRNA (800μg polymer, 50 μg siRNA) was injected into C57B1/6 mice. As in the invivo targeting studies, the siRNA conjugate was administered by tailvein infusion. Livers from injected mice were harvested two days afterinjection and assayed for apoB mRNA levels using reverse transcriptasequantitative PCR (RT-qPCR). The apoB mRNA levels were measured relativeto the level of GAPDH mRNA and μg total input RNA, in order to reducethe possibility that any differences in relative apoB mRNA levels weredue to nonspecific effects on housekeeping gene expression. As shown inFIG. 8A, mice treated with apoB-1 siRNA conjugate had significantlyreduced apoB mRNA levels compared to mice receiving a non-apoB controlsiRNA or mice injected with saline only (n=5, p<0.00001), as measuredtwo days after injection. Specifically, the mean apoB mRNA level in micereceiving apoB-1 siRNA conjugate was reduced by 76±14% compared to thesaline treated group relative to GAPDH mRNA levels, whereas apoB mRNAlevels in mice injected with the control siRNA were unaffected. Similarresults were obtained if apoB mRNA levels were measured relative tototal RNA. Western blot analysis of apoB-100 protein levels in serumreflected the reduction in liver apoB mRNA levels (FIG. 8B).

To confirm the specificity of the apoB knockdown, a separate group ofmice was treated with an siRNA targeting a different region of the apoBmRNA. Mice receiving apoB-2 siRNA conjugate also exhibited a significantreduction in apoB mRNA levels (60±6% reduction, n=5, p<0.00001, FIG.8A). Western blot analysis of apoB-100 protein levels in serum reflectedthe reduction in liver apoB mRNA levels (FIG. 8B). ApoB mRNA expressionwas not reduced in the jejunum, another tissue that expresses the apoBgene, suggesting that the conjugate did not target this tissue. Thistissue-specific activity is consistent with hepatocyte targeting.

We also prepared conjugates containing siRNAs targeting peroxisomeproliferator-activated receptor alpha (ppara), a gene important incontrolling fatty acid metabolism in liver (Kersten 1999, Schoonjans1996). Delivery of two different siRNAs targeting ppara resulted insignificant knockdown of ppara mRNA levels, as assayed by twoindependent methods for quantifying mRNA levels (FIG. 8C). Relative tomice receiving a control siRNA, ppara mRNA levels in mice receivingppara-1 siRNA were reduced by between 40±9% and 64±9%, as assayed byIINVADER® or RT-qPCR, respectively. A similar reduction in ppara mRNAlevels was observed in mice injected with delivery vehicle containingppara-2 siRNA, which targets a separate region of the ppara mRNAsequence.

The potential toxicity of the delivery system was assessed by measuringserum levels of liver enzymes and cytokines. Slight elevations of ALTand AST levels were detected in mice receiving control siRNA or apoB-1siRNA conjugates as compared to saline-treated mice, 48 h afterinjection. However, the increased levels were not significant (p<0.05)and histological examination of liver sections did not reveal signs ofliver toxicity. Similarly, analysis of TNF-α and IL-6 levels in serumusing ELISA revealed that both were slightly elevated 6 h afterinjection of siRNA-polymer conjugate, but returned to baseline by 48 h.The increases observed at 6 h would not be expected to cause significantimmune stimulation and are at least four orders of magnitude lower thanthose observed upon stimulation with lipopolysaccharide (Matsumoto 1998,Matsuzaki 2001) and one to three orders of magnitude lower than afterinjection of adenovirus (Lieber 1997, Benihoud 2007). No significantdifferences in serum levels of INF-α were detected at any of thetimepoints, except for a slight increase at 6 h after injection ofapoB-1 siRNA conjugate. These results indicate the targeted deliverysystem is well-tolerated.

Example 12 Dose-Response and Phenotypic Analysis

We performed dose-response studies in two ways: by decreasing the amountof siRNA conjugate delivered to the mice or by holding the amount ofpolymer constant but decreasing the amount of conjugated siRNA. Bycomparing the results of these experiments, we hoped to determine whichof the two components of the delivery vehicle was limiting for delivery:the endosomolytic polymer or the siRNA itself.

Injection of simple serial dilutions of the apoB-1 siRNA conjugate intomice led to a progressive decrease in the amount of knockdown of liverapoB mRNA (FIG. 9A). At the highest injected dose (800 μg polymer, 50 μgsiRNA, i.e. 2.5 mg/kg), apoB mRNA levels in the liver were reduced 84±5%relative to GAPDH mRNA on Day 2 postinjection compared to mice injectedwith saline only. Similar results were obtained when apoB mRNA levelswere measured relative to total RNA. Injection of two-fold less siRNAconjugate (400 μg polymer, 25 μg siRNA) resulted in a 50±8% reduction inrelative apoB mRNA levels. Injection of four-fold less resulted in noapoB knockdown as compared to the saline control group. Holding theamount of polymer constant but decreasing the amount of apoB-1 siRNAconjugated to the delivery vehicle led to quantitatively similar resultsto those obtained from serial dilutions (FIG. 9B). This finding suggeststhat the amount of endosomolytic polymer present in the delivery vehicleis not the limiting factor for the knockdown observed, but rather it isthe amount, or potency, of the siRNA conjugated to it.

A hallmark of apoB deficiency is decreased serum cholesterol levels dueto impairment of VLDL assembly and cholesterol transport from the liver(Burnett Zimmermann 200602). To determine if the level of knockdown ofapoB shown in FIG. 10 was sufficient to elicit a physiological responsein these mice, we measured their total serum cholesterol levels. At thehighest delivered siRNA dose (50 μg), we observed a significant decreasein mean serum cholesterol levels (30±7%, n=5, p<0.001) relative to micereceiving a control siRNA or saline only (FIG. 10). Similar results wereobtained in animals treated with apoB-2 siRNA conjugate. Decreasing theamount of siRNA delivered led to a progressive decrease in the amount ofcholesterol lowering observed, consistent with decreased apoB mRNAknockdown measured in these animals.

Impairment of VLDL assembly in the liver and the resultant decrease inVLDL export might also be expected to alter hepatic triglyceride levelsbecause triglycerides are also incorporated into VLDL particles (Gibbons2004). Indeed, transgenic mice expressing a truncated form of apoB,similar to the version found in patients with familialhypobetalipoproteinemia, also display a reduced capacity to transporthepatic triglycerides (Chen 2000). In order to assess the effects ofapoB knockdown on triglyceride transport, we performed oil red stainingof liver sections obtained from mice injected with apoB-1 siRNAconjugate. Inspection of the liver sections revealed dramaticallyincreased hepatic lipid content compared to control mice (FIG. 11).Panel A shows a mouse treated with ApoB siRNA. Panel B shows a mousetreated with negative control GL3 siRNA. Panel C shows a mouse injectedwith saline alone. Decreased serum triglyceride levels were alsodetected in these mice, providing further evidence for diminishedhepatic triglyceride export capacity. Together, these results indicatethat simple i.v. injection of apoB-1 siRNA conjugate results infunctional delivery of the polynucleotide to hepatocyte and thereforeknockdown of expression of apoB in the liver with expected phenotypiceffects.

For analysis of liver fat accumulation, liver samples fromsiRNA-conjugate-treated and control mice were frozen in OCT freezingmedium and frozen tissue sections (8-10 μm) were prepared as describedabove. Air dried tissue sections were fixed in 4% formaldehyde/PBS for20 min, rinsed in several changes of distilled water, and then rinsedbriefly with 60% isopropanol. Oil red O stock solution was prepared bymixing 0.5 g of oil red O in 100 ml of isopropanol overnight andfiltered using Whatman #1 filter papers. Oil red O working solution wasprepared by mixing 20 ml of water with 30 ml of oil red O stock solutionand filtered using a 0.2 μm Nalgene filtration unit (Fisher Scientific).The fixed sections were stained with freshly prepared oil red O workingsolution for 15 min, rinsed briefly with 60% isopropanol.Counterstaining of nuclei was performed by dipping the slides intoHarris modified hematoxylin solution (Sigma, St. Louis Mo.) 7 times andrinsed in distilled water. Rinsed slides were then mounted with GelMount (Biomedia Corp., Foster City Calif.). Stained slides were analyzedusing a Zeiss Axioplan 2 microscope equipped with a digital camera(Axiocam, Carl Zeiss). Digital images were captured using the AxioVisionsoftware (Carl Zeiss).

Example 13 Delivery of siRNA by Conjugation to Membrane Active Polymerby a CDM or Disulfide Labile Linkage

Disulfide Conjugate: 50 ng of siRNA targeted against luciferase that hada 5′-amino group on the sense strand was reacted with 1 μg ofN-succinimidyl-S-acetylthioacetate in the presence of HEPES base pH 7.5.Separately, 50 μg of a polyvinylether synthesized from a 50/50 mixtureof amine-protected and butyl vinyl ether monomers was reacted with 5 μgof 3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide ester in thepresence of HEPES pH 7.5. The modified siRNA and modified polymer werethen combined to allow covalent attachment of the siRNA to the polymer.After 6-24 h, the polymer-siRNA conjugate was reacted with 200 μg of2-propionic-3-methylmaleic anhydride in the presence of HEPES base. Theparticles were added to a mouse hepatocyte HEPA cell line that stablyexpresses the luciferase gene. As a control, samples containingCDM-modified polymer (without siRNA) were added to cells. In cellsexposed to the polymer-polynucleotide conjugate, luciferase expressionwas suppressed 75% relative to cells exposed to polymer alone,indicating delivery of functional siRNA to the cells.

CDM Conjugate: 50 ng of siRNA targeted against luciferase that had a5′-amino group on the sense strand was reacted with 2 μg of CDMthioester in the presence of HEPES pH 7.9. To the siRNA was added 50 μgof a polyvinylether synthesized from a 50/50 mixture of amine-protectedand butyl vinyl ether monomers. After 6-24 h, the polymer-siRNAconjugate was reacted with 200 μg of 2-propionic-3-methylmaleicanhydride in the presence of HEPES base. The particles were added to amouse hepatocyte HEPA cell line that stably expresses the luciferasegene. As a control, samples containing CDM-modified polymer (withoutsiRNA) were added to cells. In cells exposed to thepolymer-polynucleotide conjugate, luciferase expression was suppressed86% relative to cells exposed to polymer alone, indicating delivery offunctional siRNA to the cells.

Percent inhibition of luciferase activity following transfection ofHEPA-Luc cells using membrane active polymer-siRNA conjugates.

Disulfide Conjugation CDM Conjugation 75% 86%

Example 14 Longevity of apoB Knockdown and its Phenotypic Effect

We performed a timecourse experiment to determine the duration of apoBknockdown and cholesterol lowering in mice after injection of a singledose of apoB-1 siRNA conjugate. Consistent with our results described inthe previous sections, injection of apoB-1 siRNA conjugate (800 μgpolymer, 50 μg siRNA) resulted in reduction of mean apoB mRNA levels by87±8% on Day 2 relative to control mice (FIG. 12A). The reduction inapoB expression was accompanied by a 42±5% reduction in total serumcholesterol levels (FIG. 12B). Decreases in apoB mRNA expressionremained significant through Day 10, and had returned to near controllevels by Day 15. Reduction in serum cholesterol remained significantthrough Day 4 (n=5, p<0.01), and did not fully recover to control levelsuntil Day 10. These results indicate that sustained apoB knockdown andthe generation of the phenotype can be achieved after a single injectionof siRNA conjugate, and that the phenotype can be reversed as apoBexpression returns to normal over time.

Example 15 The Delivery of Phosphorodiamidate Morpholino Oligonucleotide(PMO) to Cells Using Polymer-PMO Conjugates

Using the CDM-thioester based crosslinking used to link siRNA andpolymer, it is also possible to reversibly conjugate other types ofoligonucleotides to polymers. In particular, we studied the delivery ofPMO to cells. 5 nmol of amino-PMO (which blocks a mutant splice site inthe mutant Luciferase transcript) either bare or hybridized with acomplimentary strand of DNA was reacted with nothing or with 2 μg of CDMthioester in the presence of HEPES pH 7.9. To this was added 200 μg ofpolyvinyl ether synthesized from 50% amino vinylether, 45% butylvinylether and 5% dodecylvinylether (DW550). After three or more hoursthe polymer+PMO or polymer-PMO conjugate was reacted with 500 μg of CDMin the presence of HEPES to maintain pH>7.5. In addition to CDM, theconjugates were reacted with PEG esters of CDM, which are derived fromthe acid chloride of CDM and PEG monomethyl ethers of various molecularweights. The conjugates were then added HeLa Luc/705 cells (Gene Tools,Philomath Oreg.) grown under conditions used for HeLa cells. The cellswere plated in 24-well culture dishes at a density of 3×10⁶ cells/welland incubated for 24 h. Media were replaced with 1.0 ml DMEM containing2.5 nmol amino-PMO complexes. The cells were incubated for 4 h in ahumidified, 5% CO₂ incubator at 37° C. The media was then replaced withDMEM containing 10% fetal bovine serum. The cells were then incubatedfor an additional 48 h. The cells were then harvested and the lysateswere then assayed for luciferase expression. The results demonstrateenhanced delivery of the uncharged polynucleotide when the transfectionagent is a DW550 polyvinylether polymer-PMO conjugate.

Delivery of Uncharged Polynucleotides to Cells

Transfection vehicle Fold induction of luciferase no linkage DW550 +bare PMO 2 DW550 + PMO hybridized with DNA 2 polymer-PMO conjugateDW550-CDM-barePMO 7.5 DW550-CDM-hybridized PMO 10

Example 16 Conjugate Delivery of siRNA to Muscle

40 μg PPARa siRNA or control (GL3) siRNA was conjugated to PBAVE polymerand masked as described above. 250 μl conjugate was injected into thegastrocnemius muscle in mice (n=3). Injection rate was about 25 μl/sec.PPARa expression levels were determined as described above.

PPARa Inhibition in Muscle Following Direct Injection of MaskedConjugate

PPARa mRNA levels relative to: siRNA conjugate GAPDH mRNA total mRNA GL31.00 ± 0.26 1.00 ± 0.15 PPARa 0.62 ± 0.07 0.54 ± 0.13

Example 17 In Vivo Delivery of siRNA Expression Cassette

20 μg of PCR-generated linear SEAP siRNA expression cassette, whichexpresses SEAP siRNA under control of the U6 promoter, was complexedwith 400 μg membrane active polymer DW1453 (composed of polymerizationof 75 mol % amino, 21 mol % lower alkyl(butyl) and 4 mol % higher alkyl(C18, octadecyl) groups and modified with galactose with lactobionicacid (LBA). Lactobionylation was performed by addition of 43 mg LBA to60 mg polymer followed by addition of 34 mg EDC and 24 mg NHS. As acontrol, a cassette expressing luciferase siRNA (GL3) was alsoformulated. The DNA was conjugated to the polymer by the addition of0.65 weight equivalents of glutaraldehyde. Following conjugationovernight, the complexes were modified with 40 mole % NHS-acetate toreduce charge on the polymer.

500 μg of lactobionylated DW1453 was modified with 7 wt equivalents ofmasking agent CDM-PEG (average 10 units) in the presence of 14 wtequivalents HEPES base and injected into the tail vein of miceexpressing secreted alkaline phosphatase (SEAP). 10 min after maskedpolymer injection, the co-targeted DNA-DW1453 polymer conjugates wereinjected. On days 1 and 14, blood was drawn and assayed for SEAP levels.

SEAP activity siRNA day 14 relative to activity at day 1 SEAP siRNA 48 ±13 GL3 siRNA 87 ± 17 Activity was the average of 4 animals ± standarddeviation.

Example 18 Delivery of Antibodies

35 μg of rabbit IGG (Sigma) antibody was modified with 2 wt % SPDP(Pierce). DW1360 (30 mg/ml in 5 mM TAPS pH 9) was modified by additionof 2 wt % iminothiolane. 530 μg of iminothiolane-PBAVE was added to 400μl isotonic glucose solution containing 5 mM TAPS pH 9. To this solutionwas added 35 μg of SPDP-modified antibody. After 16 h, theantibody-polymer conjugate was modified with 7 wt eq of a 2:1 mixture ofCDM-NAG and CDM-PEG. The conjugate was injected into the tail vein of amouse. 20 min post-injection, the mouse was sacrificed and the liver washarvested and processed for microscopic analysis. Examination of theliver tissue indicated significant accumulation of antibody inhepatocytes (FIG. 13). Upper left quadrant shows labeled antibodies,Upper right quadrant shows actin, lower left quadrant shows nuclei, andlower right quadrant shows a composite image.

Example 19 Masked Polynucleotide-Polymer Conjugate as In VitroTransfection Reagent

Primary hepatocytes were harvested from adult mice (strain C57BL/6)using the two-step collagenase perfusion procedure as previouslydescribed (Klaunig 1981). Hepatocyte viability was 85-90% as determinedby Trypan Blue exclusion. Hepatocytes were plated at a density of1.5×10⁵ cells per well in collagen coated 12-well plates in 1 ml ofmedia containing 10% fetal calf serum. 24 h after plating, cells intriplicate wells were transfected without media change with siRNA usingTRANSIT-SIQUEST® (Mirus Bio, Madison Wis.) according to themanufacturer's protocol, or with different amounts of siRNA conjugate.Hepatocytes were harvested 24 h after transfection and total RNA wasisolated with TRIREAGENT® Reagent (Molecular Research Center, CincinnatiOhio).

To demonstrate the in vitro transfection properties of the describedmasked polynucleotide-polymer conjugates, apolipoprotein B(apoB)-specific siRNA was delivered to primary hepatocytes usingcommercially available siRNA transfection reagents or the describedpolynucleotide conjugates. Transfection of the primary hepatocytes withthe conjugate was highly effective, resulting in nearly 80% knockdown ofapoB mRNA (FIG. 14). The level of siRNA delivery, as measured by targetgene knockdown was comparable to that achieved with the commercialreagent SIQUEST®. As predicted, decreasing the amount of conjugate addedto the cells led to progressively decreased apoB knockdown.

Example 20 Masked Polynucleotide-Polymer Conjugate as In VitroTransfection Reagent

Hepa-1c1c7 cells were transfected with plasmids encoding firefly andrenilla luciferases using TRANSIT LT1® (Mirus Bio). 4 h after plasmidtransfection, 100 ng luciferase siRNA conjugated with 9 μg PD-PLLfollowed by modification with CDM-Pip-LBA (45 g) was added to cells. Forsome sample, CDM-Pip-LBA modified 25/75-PBAVE (5 μg) was also added tocells. 48 after addition of siRNA, the cells were harvested and assayedfor firefly and renilla luciferase. The amount of firefly expression wasnormalized to renilla for each sample and to cells not exposed to siRNAfor each group. Alone, PLL-siRNA conjugates are unable to deliver siRNAto cells in vitro (white bar, FIG. 15). Addition of maleamate-masked,membrane lytic polymer PBAVE results in activity equal to commercialtransfection reagent (black bar). In general, siRNA and DNAtransfections appear to require excess reagent. Excess releasingpolymer, above that needed to interact with siRNA, may enhance in vitroand in vivo functional delivery (release from endosomes). Due to thesimilar size and charge between siRNA-polymer conjugates and polymers,they can simultaneously target hepatocytes in vivo. In support of thisexpectation, we observed that coinjection of Cy3-labeled siRNAconjugated to PLL with Oregon Green-labeled 25/75-PBAVE did indeedcolocalize to hepatocytes (see above).

The in vitro activity of the siRNA conjugate with delivery polymers isequal to unconjugated siRNA transfected with commercial reagent TRANSITTKO® (FIG. 15). Experiments in parallel with an irreversible linkage ofsiRNA and polymer based upon iodoacetamide alkylation chemistry(IA-conjugate spotted bar in FIG. 15) demonstrated that siRNA-polymerconjugates are not active if the conjugation is not reversible,suggesting that the disulfide bond must be reduced in order forfunctional delivery of siRNA (The 20% knockdown observed is due to theroughly 10% of oligo that was not conjugated, as determined by gelanalysis). The activity observed for the maleamylated conjugatessuggests that, even though the polymer is no longer positively-chargedand interacting with the oligonucleotide by electrostatic forces, thesiRNA-conjugate is resistant to degradation by nucleases in the serum.Modified siRNA analogues that are nuclease resistant may also be used.

Example 21 Labile Linkages for Attachment of Masking Agents andPolynucleotides to a Membrane Active Polymer

A. pH labile bond with PEG spacer. One embodiment of an ASGP-R ligandtarget group may be prepared as follows:

Compound I was used as a precursor to create compound II, a galactosetargeting group masking agent capable of reversibly modifyingamine-containing membrane active polymers such as PBAVE polymers.Reaction of the maleic anhydride with an anime group on the polymersresults in formation of a pH labile linkage between the galactose andthe polymer. Modification of a PBAVE polymer with compound II resultsin:

Modification of PBAVE amino groups with compound III (0.75:1 compoundIII to polymer amine molar ratio) and CDM-PEG₅₀₀ (0.25 CDM-PEG topolymer amine molar ratio) resulted in a conjugate which exhibitedtargeting to hepatocytes in vivo. Varying spacer lengths of ethylenegroups (n=1, 2, 3, or 7) were all effective in delivering siRNA tohepatocytes in vivo as evidenced by knock down of target geneexpression.

B. pH labile bond with Alkyl spacer. Alkyl spacer groups may also beused as illustrated in compound V. Alkyl spacers may, however, increasethe hydrophobicity of the masking agent, resulting in lower solubilityof the masking agent and the conjugate.

C. Multivalent targeting groups. Targeting groups with higher valencymay also be utilized. Exemplary embodiments of a multivalent galactosetargeting groups are illustrated below. Divalent (VI) and trivalent(VII) galactose ligands may have higher affinity for the ASGP-R.

D. Negative control targeting ligands. Related targeting groups whichhave significantly lower affinity for the target cell may be used asnegative controls to determine efficacy of the preferred targetinggroup. As an example, the ASGP-R of hepatocyte binds galactose, but notglucose. Therefore, a glucose targeting groups (VIII) may serve as anegative control in cell targeting assays.

E. Mannose targeting group. Macrophages and liver Kupffer cells areknown to have receptors which bind mannose saccharides. Therefore,targeting groups with mannose residues (IX) may be used to target thesecells.

F. Linkages with varying lability. Linkages of varying lability(kinetics of cleavage) can be used to connect the masking agent orpolynucleotide to the membrane active polymer.

For delivery of antisense polynucleotides, it may be possible toincrease the duration of knockdown by decreasing the rate ofpolynucleotide release from the conjugate. Disulfide bonds can be madewith varying kinetics of cleavage in the reducing environment in atypical mammalian cell. A slower release of masking agent form thepolymer may increase circulation time of the conjugate in vivo. Forexample, 5-methyl-2-iminothiolane (M2IT, Linkage X) exhibits a 4× slowerrate of cleavage than an SMTP linkage (Compound XI).

The cleavage rate for Compound XII is expected to be about 30× slowerthan for disubstituted maleic anhydride linkages.

Example 22 Characterization of PBA VE and Polynucleotide-PBA VEConjugates

A. Amphipathic analysis. 1,6-diphenyl-1,3,5-hexatriene (DPH, Invitrogen)fluorescence (λ_(ex)=350 nm; λ_(em)=452 nm) is enhanced in a hydrophobicenvironment. This fluorophore was used to analyze the PBAVE (DW1360)polymer. 0.5 μM (final concentration) DPH was added to 10 μg PBAVE inthe presence or absence of 40 μg plasmid DNA in 0.5 ml 50 mM HEPESbuffer, pH 8.0. The solution was then tested for DPH accumulation in ahydrophobic environment by measuring fluorescence of DPH. Increase DPHfluorescence in the presence of the conjugates indicates the formationof a hydrophobic environment by the polymer. Addition of excess DNA didnot significantly alter DPH fluorescence (FIG. 16).

B. Molecular Weight. The molecular weight of DW1360 was determined usingHPLC size exclusion chromatography and a set of polyethyleneglycols asstandards (American Polymer Standard Corp.). The polymers were separatedon a GPC HPLC using a Shodec SB-803 HQ column with 0.2 M LiClO₄, 5% AcOHin methanol. Elution of the PBAVE polymer from the column indicated asize average of about 12.8 kDa (FIG. 17)

C. Molecular weight of polynucleotide-polymer conjugate. The molecularweight of masked polynucleotide-polymer conjugate was determined usingFPLC size exclusion chromatography and Cy3-labeled nucleic acids asstandards. The conjugate and standards were separated on a Shimadzu FPLCat using a 1×22 cm Sephacryl S-500 column and eluted with 50 mM HEPES,pH 8.0 at 1 ml/min. Elution of the conjugate from the column indicated asize average of about 77 kDa (FIG. 18).

D. Conjugate stoichiometry. The average molecular weight of the PBAVEpolymer was experimentally determined to be about 13 kDa. The averagemolecular weight of the masked polynucleotide-polymer conjugate wasexperimentally determined to be about 77 kDa. The average moleculeweight per charge (amine) of the PBAVE polymer was experimentallydetermined to be about 200 Da. The average molecular weight of the CDMmasking agent was calculated to be about 550 Da. The average degree ofmodification of the PBAVE polymer was experimentally determined to beabout 75% of available amines. The average molecular weight of theconjugated siRNA was calculated to be about 14 kDa. Using these values,the average molecular weight of the CDM-masked PBAVE polymer maskingagent was calculated to be about 30 kDa. Thus, the likely stoichiometryof siRNA to masked polymer was estimated to be about 1:2.

E. Particle Sizing and Zeta Potential. The sizes of the conjugatesinjected in vivo were measured by light scattering at 532 nm using aBrookhaven Instruments Corporation, ZetaPlus Particle Sizer, I90.Unimodal analysis of peaks varied between 100 and 30 nm. Usingmultimodal analysis, the peak with the largest number (>95%) ofparticles were less than 50 nm and more typically less than 20 nm.Polyconjugate size can also be measured using analyticalultracentrifugation or atomic force microscopy. In the same way the zetapotential of the conjugates were measured using Brookhaven InstrumentsCorporation ZetaPALS. The zeta potential of the CDM-masked conjugatesvaried between 0 and −30 mV and more predominantly between 0 and −20 mV.Zeta potential was measure in isotonic glucose buffered at pH9 with 5 mMTAPS. Stable, non-aggregating conjugates were also formed with zetapotentials between 0 and −10 mV and between 0 and −5 mV under the sameconditions. At pH 7, the conjugates would be expected to gain somepositive charge due to protonation of some of the amines.

Example 23 In Vivo Delivery of Conjugate to Human Hepatoma Xenograft inNude Mice

To demonstrate that the described conjugates can be utilized to targettumor cells CDM-NAG modified oligonucleotide-DW1360 conjugate wasinjected into mice that had been implanted with human hepatocarcinomaHepG2 cells subcutaneously (SC) on the flank. Six week old femaleathymic nude mice were obtained from Harlan Spraque Dawley(Indianapolis, Ind.). Mice were inoculated with 2 million HepG2 cells in300 μl PBS subcutaneously on the left flank. Control colon carcinomaHT-29 cells (2 million in 300 μl of PBS) were inoculated SC on the rightflank 2 weeks later. Later injection was to compensate for faster growthrate. When SC tumors grew to approximately 8 mm in width and length,CDM-NAG and CDM-PEG modified conjugate containing 10 μg Cy3-labeled21-mer dsDNA in 200 μl delivery buffer was injected via the tail vein.

Accumulation of NAG targeted conjugate was observed in a largepercentage of the SC hepatoma tumor mass (30-60%, n=4, FIG. 19A).Representative area of tumor cells showing uptake is shown in FIG. 19A.A very low level of tumor targeting was observed in mice receivingglucose targeted conjugate (FIG. 19B, <5% of tumor mass, n=3). Incontrol colon carcinoma tumors, without galactose receptor, no tumorcell signal was observed with either NAG or glucose modified conjugate(FIG. 19 C-D, n=2). Similar results were also observed using HuH-7hepatocarcinoma cells to establish xenografts.

Example 24 Modification of Polymer with Reversible PEG ModificationPrior to Conjugation

400 μg of polymer DW1360 (formed from a feed ratio of 75:20:5amine:butyl:octadecyl vinyl ethers) was modified with 1.5 wt % SMPT. 1 hlater 2 wt equivalents of a 2:1 wt:wt mixture of CDM-NAG(N-acetylgalactoseamine) and CDM-PEG (average 11 unites) was added tothe polymer in the presence of 5.6 mg of HEPES base. To this solutionwas added 20 μg of SATA/Cy3-modified 22-mer DNA oligonucleotides. Afterovernight incubation, 5 wt equivalents of a 2:1 wt:wt mixture of CDM-NAGand CDM-PEG was added to the conjugate. The masked conjugate wasinjected in 400 μl of saline into the tail vein of a mouse. It wasdetermined that the modification of polymer with CDM-NAG and CDM-PEGprior to conjugation did not reduce the conjugation efficiency. 1 hpostinjection, the liver was harvested and frozen liver sections wereprepared and processed for immunohistochemistry. TO-PRO-3® was used tostain cell nuclei. After injection of into the tail vein of mice,labeled oligonucleotide was observed in hepatocytes.

Example 25 Quantification of Amine Groups in Conjugate after CDM-ReagentModification

Oligonucleotide-DW 1360 conjugate polymer was synthesized as describedpreviously followed by treatment with 14 wt equivalents HEPES base and 7wt equivalents of a 2:1 wt:wt mixture of CDM-NAG and CDM-PEG (average 11units). One hour later, the amine content of the maleic anhydridederivative treated conjugate was measured by treatment withtrinitrobenzene sulfonic acid (TNBS) in 100 mM NaHCO₃. When normalizedto a conjugate that had not been maleamate modified, it was determinedthat the amount of modified amines was about 75% of total. This degreeof modification may be varied by changing the amount of added maleicanhydride.

Example 26 Electrophoresis of Oligonucleotide-Polymer Conjugate and itsMaleic Anhydride Derivatives

Oligonucleotide-polymer DW1360 conjugates were made as describedpreviously. The conjugate containing 1 μg of oligonucleotide was thenmodified with varying amounts of CDM-PEG (11 units average) and CDM-NAG.The conjugates were then placed into agarose gels (2 wt %) andelectrophoresed. It was noted upon staining with ethidium bromide thatthe conjugates' charge was altered depending on the equivalents ofmaleic anhydride derivate from positive to neutral to negative.

The foregoing is considered as illustrative only of the principles ofthe invention. Furthermore, since numerous modifications and changeswill readily occur to those skilled in the art, it is not desired tolimit the invention to the exact construction and operation shown anddescribed. Therefore, all suitable modifications and equivalents fallwithin the scope of the invention.

We claim:
 1. A conjugate delivery system for delivering an RNAinterference polynucleotide to a liver cell in vivo comprising:

wherein, P is a membrane active polyamine, L² is a disubstitutedmaleamate linkage, M¹ is a masking agent containing a galactosederivative, M² is a masking agent containing a polyethyleneglycol, N isan RNA interference polynucleotide, L¹ is a physiologically labilecovalent linkage that is orthogonal to L², y and z are integers greaterthan 1 wherein the value of y and z together is greater than 50% onamines of P, positive charge of P is neutralized and membrane activityof P is reversibly inhibited by modification of greater than 50% of Pamines by attachment of M¹ and M² through the physiologically pH-labilelinkages L², and cleavage of L² restores membrane activity of P.
 2. Theconjugate of claim 1 wherein the polynucleotide comprises siRNA ormicroRNA.
 3. The conjugate of claim 1 wherein the polyamine is largerthan 5 kDa.
 4. The conjugate of claim 3 wherein the polyamine is largerthan 7.5 kDa.
 5. The conjugate of claim 4 wherein the polymer is largerthan 10 kDa.
 6. The conjugate of claim 1 wherein the conjugate has azeta potential between 0 and −20 mV at pH
 9. 7. The conjugate of claim 6wherein the conjugate has a zeta potential between 0 and −10 mV at pH 9.8. The conjugate of claim 7 wherein the conjugate has a zeta potentialbetween 0 and −5 mV at pH
 9. 9. The conjugate of claim 1 wherein P-L²-M¹has the structure:

wherein n is an integer between 1 and
 500. 10. The conjugate of claim 1wherein P-L²-M¹ has the structure:

wherein n is an integer between 1 and
 500. 11. The conjugate of claim 1wherein the conjugate is smaller than 20 nm in diameter.
 12. Theconjugate of claim 1 wherein the conjugate is smaller than 10 nm indiameter.
 13. The conjugate of claim 1 wherein the membrane activepolymer consists of a polyvinylether random copolymer.
 14. The conjugateof claim 1 wherein the membrane active polymer contains two or moredifferent monomers.
 15. The conjugate of claim 14 wherein the membraneactive polymer contains three or more different monomers.
 16. Theconjugate of claim 15 wherein the monomers are selected from the groupconsisting of: amine-containing monomers, lower alkyl monomers, butylmonomers, higher alkyl monomers, dodecyl monomers, and octadecylmonomers.
 17. The conjugate of claim 13 wherein the polyvinyletherrandom copolymer is composed of amine-containing vinyl ether monomers,lower alkyl vinyl ether monomers, and higher alkyl vinyl ether monomers.18. The conjugate of claim 17 wherein the lower alkyl vinyl etherconsists of a butyl vinyl ether and the higher alkyl vinyl etherconsists of an octadecyl vinyl ether or dodecyl vinyl ether.
 19. Theconjugate of claim 18 wherein the polyvinylether random copolymer issoluble in water.
 20. The conjugate of claim 18 wherein thepolyvinylether random copolymer is surface active.
 21. The conjugate ofclaim 1 wherein the polymer is a biodegradable polymer.
 22. Theconjugate of claim 1 wherein the masking agents are reversibly linked toat least 70% of the amines on the polyamine.
 23. The conjugate of claim17 wherein the amine-containing vinyl ether monomers, lower alkyl vinylether monomers, and higher alkyl vinyl ether monomers are present in aratio of 5.4-7.5 a amine-containing vinyl ether monomers:3-3.5 loweralkyl vinyl ether monomers:1 higher alkyl vinyl ether monomers.
 24. Theconjugate delivery system of claim 1 wherein the conjugate deliverysystem further comprises an excess of membrane active polyaminereversibly covalently linked to a plurality of masking agents viaphysiologically labile bonds wherein the excess polymer is notcovalently linked to the RNA interference polynucleotide.
 25. Theconjugate delivery system of claim 1 wherein the conjugate is providedin a pharmaceutically acceptable carrier or diluent.
 26. The conjugateof claim 2 wherein the polynucleotide is selected from the groupsconsisting of: DNA, RNA, dsRNA, RNA interference polynucleotide, siRNA,and miRNA.
 27. A conjugate delivery system for delivering an RNAinterference polynucleotide to a liver cell in vivo formed by theprocess comprising: a) forming a membrane active polyamine; b) forming afirst masking agent comprising a disubstituted maleic anhydridecontaining a galactose derivative; c) forming a second masking agentcomprising a disubstituted maleic anhydride containing apolyethyleneglycol; d) linking the RNA interference polynucleotide tothe polyamine via a physiologically labile covalent bond that isorthogonal to a maleamate linkage; e) reversibly inhibiting membraneactivity of the membrane active polyamine wherein the inhibitingconsists of modifying 50% or more of the amines on the polyamine byreacting the polyamine with the first and second masking agents therebylinking a plurality of galactose derivatives and a plurality ofpolyethyleneglycols to the membrane active polymer via physiologicallypH-labile disubstituted maleamate linkages.